The present invention relates to nucleic acid probes derived from the spacer region between the 16S and 23S ribosomal ribonucleic acid (rRNA) genes, to be used for the specific detection of eubacterial organisms in, a biological sample by a hybridization procedure, as well as to nucleic acid primers to be used for the amplification of said spacer region of eubacterial organisms in a biological sample. The present invention also relates to new spacer region sequences from which said probes or primers may be derived.
Since the advent of the polymerase chain reaction and some other nucleic acid amplification techniques the impact of DNA-probe technology in the diagnosis of micro-organisms in biological samples of all sorts is increasing. Being often more specific and potentially more sensitivexe2x80x94if an adequate amplification and or detection system is used the DNA probe approach may eventually replace the conventional identification techniques.
The reliability of nucleic acid based tests essentially depends on the sensitivity and specificaty of the probes and/or primers used. Thus the corner stone of this type of assay is the identification of nucleic acid sequences which are unique to the group of organisms of interest.
Most of the nucleic acid based tests either described in literature and/or commercially available aim at the detection of just one particular organism in a biological sample, Since most biological samples usually may contain a great variety of clinically relevant micro-organisms, a multitude of separate assays have to be performed to detect all relevant organisms possibly present. This approach would be very expensive, laborious and time-consuming. Consequently, the number of tests actually performed in most routine diagnostic labs on a particular sample is restricted to the detection of just a few of the most relevant organisms. Therefore it would be extremely convenient to have access to a system which enables the fast, easy and simultaneous detection of a multitude of different organisms. The more organisms that can be screened for in the same assay, the more cost-effective the procedure would be.
As put forward in earlier published documents, the spacer region situated between the 16S rRNA and the 23S rRNA gene, also referred to as the internal transcribed spacer (ITS), is an advantageous target region for probe development for detection of pathogens of bacterial origin (International application WO 91/16454; Rossau et al., 1992; EP-A-0 395 292).
One of its most appreciated advantages, is that sequences unique to a great variety of bacterial taxa can be found in a very limited area of the bacterial genome. This characteristic allows for an advantageous design of xe2x80x9cprobe-panelsxe2x80x9d enabling the simultaneous detection of a set of organisms possibly present in a particular type of a biological sample. Moreover, being flanked by quasi-universally conserved nucleotide sequencesxe2x80x94more particularly located in the 3xe2x80x2-part of the 16S rRNA gene and the 5xe2x80x2-part of the 23S rRNA gene respectivelyxe2x80x94almost all spacers can be simultaneously amplified with a limited set of amplification primers. Alternatively, specific primer sets can be derived from the spacer sequences themselves, thereby allowing species- or group-specific amplifications.
The 16S-23S rRNA spacer region is a relatively short (about 200 to 1000 base pairs) stretch of DNA present in one or multiple copies in the genome of almost all eubacterial organisms. If multiple copies are present in the genome of one bacterium these copies can either be identical (as is most probably the case in some Neisseria species) or may differ from each other (as is the case for E. coli). This difference can be limited to a few nucleotides but also deletions and insertions of considerable length may be present.
Uptil now, spacer probes are only described and made publicly available for a limited number of organisms many of which were disclosed in international application WO 91/16454. As described above, it would be very advantageous to be able to detect simultaneously a panel of pathogens: e.g. a panel of pathogens possibly present in the same type of biological sample, or a panel of pathogens possibly causing the same type of disease symptoms, which are difficult to differentiate clinically and/or biochemically, or a panel of organisms belonging to the same taxon. In order to make the different panels as complete as possible, additional probes or sets of probes located in the spacer region and enabling the identification of at least the following bacterial groups or species are required:
Mycobacterium species
Listeria species
Chlamydia species
Acinetobacter species
Mycoplasma species
Streptococcus species
Staphylococcus species
Salmonella species
Brucella species
Yersinia species
Pseudomonas species
These additional spacer probes need to be meticulously designed such that they can be used simultaneously with at least one other probe, under the same hybridization and wash conditions, allowing the detection of a particular panel of organisms.
It is thus the aim of the present invention to select probes or sets of probes, which have as target the 16S-23S rRNA spacer region, and which allow the detection and identification of at least one, and preferably more than one, of the above mentioned micro-organisms. The probes or probe sets are selected in such a way that they can be used in combination with at least one other probe, preferably also originating from the 16S-23S rRNA spacer region, under the same hybridization and wash conditions, to allow possibly the simultaneous detection of several micro-organisms in a sample.
It is also an aim of the present invention to provide for a selection method for use in the selection of said spacer probes or probe sets.
It is also an aim of the present invention to provide a rapid and reliable hybridization method for detection and identification of at least one micro-organism in a sample, or for the simultaneous detection and identification of several micro-organisms in a sample.
It is more particularly an aim of the present invention to provide a hybridization method allowing simultaneous detection and identification of a set of micro-organisms, liable to be present in a particular type of sample.
It is more particularly an aim of the present invention to provide probes or sets of probes for the possible simultaneous detection of micro-organisms in a sample originating from respiratory tract.
It is another particular aim of the present invention to provide probes or sets of probes for the possible simultaneous detection of micro-organisms in a sample originating from cerebrospinal fluid.
It is still another particular aim of the present invention to provide probes or sets of probes for the possible simultaneous detection of micro-organisms in a sample originating from urogenital tract.
It is still another particular aim of the present invention to provide probes or sets of probes for the possible simultaneous detection of micro-organisms in a sample taken from the gastro-intestinal tract of a patient.
It is still another particular aim of the present invention to provide probes or sets of probes for the possible simultaneous detection of micro-organisms in a sample originating from food or environmental samples.
It is moreover an aim of the present invention to provide a method for detection and identification of a particular taxon in a sample, or a set of particular taxa, said taxon being either a complete genus, or a subgroup within a genus, a species or even subtypes within a species (subspecies, serovars, sequevars, biovars . . . ).
It is more particularly an aim of the present invention to provide probes or sets of probes for the detection of Mycobacterium species and subspecies, more particularly for the detection of M. tuberculosis complex strains. Mycobacterium strains from the MAIS-complex, M. avium and M. paratuberculosis, M. intracellulare and M. intracellulare-like strains, M. scrofulaceum, M. kansasii, M. chelonae, M. gordonae, M. ulcerans, M. genavense, M. xenopi, M. simiae, M. fortuitum, M. malmoense, M. celatum and M. haemophilum. 
It is also an aim of the present invention to provide probes or sets of probes for the detection of Mycoplasma strains, more particularly of M. pneumoniae and M. genitalium. 
It is also an aim of the present invention to provide probes or sets of probes for the detection of Pseudomonal strains, more particularly P. aeruginosa. 
It is also an aim of the present invention to provide probes or sets of probes for detection of Staphylococcus species, more particularly S. aureus and S. epidermidis. 
It is also an aim of the present invention to provide probes or sets of probes for the detection of Acinetobacter strains, more particularly A. baumanii. 
It is also an aim of the present invention to provide probes or sets of probes for the detection of Listeria strains, more particularly Listeria monocytogenes. 
It is also an aim of the present invention to provide probes or sets of probes for the detection of Brucella strains.
It is also an aim of the present invention to provide probes or sets of probes for the detection of Salmonella strains.
It is also an aim of the present invention to provide probes or sets of probes for the detection of Chlamydia strains, more particularly C. trachomatis and C. psittaci. 
It is also an aim of the present invention to provide probes or sets of probes for the detection of Streptococcus strains.
It is also an aim of the present invention to provide probes or sets of probes for the detection of Yersinia enterolitica strains.
It is also an aim of the present invention to provide primers allowing specific amplification of the 16S-23S rRNA spacer region for certain organisms. More particularly, it is an aim of the present invention to provide primers for the specific amplification of the spacer region of Mycobacterium, Chlamydia, Listeria, Brucella and Yersinia enterolitica strains.
It is also an aim of the present invention to provide new sequences of 16S-23S rRNA spacer regions from which useful spacer probes or primes can be derived.
It is also an aim of the present invention to provide for kits for detection of at least one organism in a sample in which said probes and/or primers are used.
It is noted that for a few of the above-mentioned organisms spacer sequences have already been published in literature or in publicly accessable data-banks.
However, it should be made clear that the spacer region sequences disclosed in the current invention (FIGS. 1-103) are new and, in case they originate from the same species as those of which a spacer sequence was already described in the prior art, they differ to some extent from the already described sequences.
Moreover, it is the principal aim of the present invention to select, from the compilation of sequence data on spacer regions, specific probes and sets of probes enabling the detection and identification of a particular panel of organisms, be it the organisms belonging to a common taxon, or the organism possibly present in the same type of sample.
The selection procedure usually consists of a theoretical and an experimental part. First of all, the different spacer sequences need to be aligned to those of the xe2x80x98closest neighboursxe2x80x99 or to the spacer sequences of other micro-organisms liable to be present in the same sample. This requires of course the sequence determination of the spacer region, as described in the examples. From the alignment, regions of divergence can be defined, from which probes with desired hybridization characteristics are designed, according to guidelines known to the man skilled in the art and specified in more detail below,
Secondly, the designed probes need to be tested experimentally and evaluated for their usefulness under specific hybridization conditions and/or in combination with other probes. Experimental tasting can be done according to any hybridization method known in the art, but a preferred assay for the simultaneous testing of different probes under the same conditions is the reverse hybridization assay. A specific format for reverse hybridization of different probes simultaneously used in the current invention is the LiPA (Line Probe Assay) as described below.
Upon experimental testing unexpected hybridization behaviour may show up when the probes are hybridized to the target nucleic acid, and specific probe adaptations may be required.
Moreover, specificity and sensitivity of the probes need to be tested with a large collection of straits, both belonging to the taxon to be detected and belonging to other ma. Due to genome heterogeneity in the spacer region, or the existence of multiple spacer regions with different sequences in the same organisms, it is quite often necessary to sequence spacer regions of additional strains, or to sequence additional spacer regions in the same strain, and redesign the probes according to the new sequence data in order to obtain a better sensitivity and/or specificity (see e.g. example 3). In some cases it may be necessary or preferable to use several probes for the same organism (see e.g. example 2 and 7). Also, upon sequencing the spacer region, some organisms may show unexpected (un)relatedness, which may lead to a revision of strain classification contrary to classical taxonomic criteria (see e.g. examples 2 and 7).
In conclusion, the experimental part of the probe selection procedure is indispensable and complementary to the theoretical part. Probe design, especially under the fixed conditions of reverse hybridization (the same conditions for each probe) is not straightforward and probes have to be evaluated meticulously before they can be used in a reverse hybridization format. Therefor, probes cannot always be simply derived on a theoretical basis from a known gene sequence.
For designing probes with desired characteristics the following useful guidelines may be followed.
Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions, explained further herein, are known to those skilled in the art.
First, the stability of the [probe:target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long A and T rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and % GC result in a Tm about 2-10xc2x0 C. higher than the temperature at which the final assay will be performed. The base composition of the probe is significant because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be stable at higher temperatures.
Conditions such as ionic strength and incubation temperature under which a probe will be used should also be taken into account in constructing a probe. It is known that hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5xc2x0 C. below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity.
It is desirable to have probes which hybridize only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency is chosen to maximize the difference in stability between the hybrid formed with the target and the nontarget nucleic acid. In some examples of the current invention, e.g. when highly related organisms need to be differentiated, it may be necessary to detect single base pair changes. In those cases, conditions of very high stringency are needed.
Second, probes should be positioned so as to minimize the stability of the [probe:nontarget] nucleic acid hybrid. This may be accomplished by minimizing the length of perfect complementarity to non-target organisms, avoiding CC rich regions of homology to non-target sequences, and by positioning the probe to span as many destabilizing mismatches as possible. Whether a probe sequence is useful to detect only a specific type of organism depends largely on the thermal stability difference between [probe:target] hybrids and [probe:nontarget] hybrids. In designing probes, the differences in these Tm values should be as large as possible (e.g. at least 2xc2x0 C. and preferably 5xc2x0 C.),
The length of the caret nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another which differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly complementary base sequence will normally primarily determine hybrid stability. While oligonucleotide probes of different lengths and base composition may be used, oligonucleotide probes preferred in this invention are between about 10 to 50 bases in length and are sufficiently homologous to the target nucleic acid.
Third, regions in the target DNA or RNA which are known to form strong internal structures inhibitory to hybridization are less preferred. Likewise, probes with extensive self-complementarity should be avoided. As explained above, hybridization is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. There can be intermolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. Computer programs are available to search for this type of interaction. However, in certain instances, it may not be possible to avoid this type of interaction.
The probes of the present invention are designed for attaining optimal performance under the same hybridization conditions so that they can be used in sets for simultaneous hybridization; this highly increases the usability of these probes and results in a significant gain in time and labour. Evidently, when other hybridization conditions should be preferred, all probes should be adapted accordingly by adding or deleting a number of nucleotides at their extremities. It should be understood that these concommitant adaptations should give rise to essentially the same result, namely that the respective probes still hybridize specifically with the defined target. Such adaptations might also be necessary if the amplified material should be RNA in nature and not DNA as in the case for the NASBA system.
The hybridization conditions can be monitored relying upon several parameters, such as the nature and concentration of the components of the media, and the temperatures under which the hybrids are formed and washed.
The hybridization and wash temperature is limited in upper value depending on the sequence of the probe (its nucleic acid composition, kind and length). The maximum hybridization or wash temperature of the probes described to the present invention ranges from 40xc2x0 C. to 60xc2x0 C., more preferably from 45xc2x0 C. to 55xc2x0 C., in the specific hybridization and wash media as described in the Examples section. At higher temperatures duolexing (=formation of the hybrids) competes with the dissociation (or denaturation) of the hybrid formed between the probe and the target.
In a preferred hybridization medium of the invention, containing 3xc3x97SSC and 20% formamide, hybridization temperances can range from 45xc2x0 C. to 55xc2x0 C., with a preferred hybridization temperature of 50xc2x0 C. A preferred wash medium contains 3xc3x97SSC and 20% formamide, and preferred wash temperatures are the same as the preferred hybridization temperatures, i.e. preferably between 45xc2x0 C, and 55xc2x0 C., and most preferably 50xc2x0 C.
However, when modifications are introduced, be it either in the probes or in the media, the temperatures at which the probes can be used to obtain the required specity should be changed according to known relationships, such as those described in the following reference: Hames B and Higgins S (eds.). Nucleic acid hybridization. A practical approach, IRL Press, Oxford, U.K., 1985.
The selected nucleic acid probes derived from the 16S-23S rRNA spacer region and described by the present invention are listed in Table 1a (SEQ ID NO 1 to 64, 175 to 191, 193 to 201, and 210 to 212). As described in the examples section, some of these probes show a better sensitivity and/or specificity than others, and the better probes are therefore preferentially used in methods to detect the organism of interest in a biological sample. However, it is possible that for certain applications (e.g. epidemiology, substrata typing, . . . ) a set of probes including the less specific and/or less sensitive probes may be very informative (see e.g. example 7).
The following definitions serve to illustrate the terms and expressions used in the different embodiments of the present Invention as set out below.
The term xe2x80x9cspacerxe2x80x9d is an abbreviated term referring to the 16S-23S rRNA internal transcribed spacer region.
The term xe2x80x9cprobexe2x80x9d refers to single stranded sequence-specific oligonucleotides which have a sequence which is sufficiently complementary to hybridize to the target sequence to be detected.
The more specific term xe2x80x9cspacer probexe2x80x9d refers to a probe as defined above having a sequence which is sufficiently complementary to hybridize to a target sequence which is located in the spacer region(s) of the organism (or group of organisms) to be detected.
Preferably said probes are 70%, 80%, 90%, or more than 95% homologous to the exact complement of the target sequence to be detected. These target sequences are either genomic DNA or precursor RNA, or amplified versions thereof.
Preferably, these probes are about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides. The nucleotides as used in the present invention may be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridization characteristics. Moreover, it is obvious to the man skilled in the art that any of the below-specified probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U).
The probes according to the invention can be formed by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be by cleaving the latter out from the closed plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes according to the presets invention can also be synthesized chemically, for instance by the conventional phospho-triester method.
The term xe2x80x9ccomplementaryxe2x80x9d nucleic acids as used herein means that the nucleic acid sequences can form a perfect base-paired double helix with each other.
The term xe2x80x9chomologousxe2x80x9d as used in the current application is synonymous for identical: this means that polynucleic acids which are said to be e.g. 80% homologous show 80% identical base pairs in the same position upon alignment of the sequences.
The term xe2x80x9cpolynucleic acidxe2x80x9d corresponds to either double-stranded or single stranded cDNA or genamic DNA or RNA, containing at least 10, 20, 30, 40 or 50 contiguous nucleotides. A polynucleic acid which is smaller than 100 nucleotides in length is often also referred to as an oligonucleotide. Single stranded polynucleic acid sequences are always represented in the current invention from the 5xe2x80x2 end to the 3xe2x80x2 end.
The term xe2x80x9cclosest neighbourxe2x80x9d means the taxon which is known or expected to be most closely related in terms of DNA homology and which has to be differentiated from the organism of interest.
The expression xe2x80x9cdesired hybridization characteristicsxe2x80x9d means that the probe only hybridizes to the DNA or RNA from organisms for which it was designed, and not to DNA or RNA from other organisms (closest neighbours or organisms liable to be present in the same sample). In practice, this means that the intensity of the hybridization signal is at least two, three, four, five, ten or more times stronger with the target DNA or RNA from the organisms for which the probes were designed, as compared to non-target sequences.
These desired hybridization characteristics correspond to what is called later in the text xe2x80x9cspecific hybridizationxe2x80x9d.
The expression xe2x80x9ctaxon-specific hybridizationxe2x80x9d or xe2x80x9ctaxon-specific probexe2x80x9d means that the probe only hybridizes to the DNA or RNA from the taxon for which it was designed and not to DNA or RNA from other taxa.
The term taxon can refer to a complete genus or a sub-group within a genus, a species or even subtype within a species (subspecies, serovars, sequevars, biovars, . . . ).
The term xe2x80x9cspecific amplificationxe2x80x9d or xe2x80x9cspecific primersxe2x80x9d refers to the fact that said primers only amplify the spacer region from these organisms for which they were designed, and not from other organisms.
The term xe2x80x9csensitivityxe2x80x9d refers to the number of false negatives: i.e. if 1 of the 100 strains to be detected is missed out, the test shows a sensitivity of (100xe2x88x921/100)%=99%.
The term xe2x80x9cspecificityxe2x80x9d refers to the number of false positives; i.e. if on 100 strains detected, 2 seem to belong to organisms for which the test is not designed, the specificity of the test is (100xe2x88x922/100)%=98%.
The probes selected as being xe2x80x9cpreferentialxe2x80x9d show a sensitivity and specificity of more than 80%, preferably more than 90% and most preferably more than 95%.
The term xe2x80x9cprimerxe2x80x9d refers to a single stranded DNA oligonucleotide sequence capable of acting as a point or initiation for synthesis or a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength. The fact that amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification is amply documented in the literature (Kwok et al., 1990).
The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988: Wu and Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton. 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of Qxcex2 replicate (Lizardi et al., 1988; Lomeii et al., 1989) or any other suitable method to amplify nucleic acid molecules known in the art.
The oligonucleotides used as primers or probes may also comprise nucleotide analogues such as phosphorothioares (Matsukura et al., 1987), alkylphosphorothioates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984).
As most other variations or modifications introduced into the original DNA sequences of the invention these variations will necessitate adaptions with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However the eventual results of hybridization will be essentially the same as those obtained with the unmodified oligonucleotides.
The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotide molecules, etc.
The term xe2x80x9csolid supportxe2x80x9d can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics acid provided that the background level of hybridization remains low. Usually the solid substrate will be a microliter plate, a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead). Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fixation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups. NH2 groups, SH groups, carboxylic groups, or coupling with biotin, haptens or proteins.
The term xe2x80x9clabelledxe2x80x9d refers to the use of labelled nucleic acids. Labelling may be carried out by the use of labelled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or by the use of labelled primers, or by any other method known to the person skilled in the art. The nature of the label may be isotopic (32P, 35S, etc.) or non-isotopic (biotin, digoxigenin, etc.).
The xe2x80x9csamplexe2x80x9d may be any biological material taken either directly from the infected human being (or animal), or after culturing (enrichment), or a sample taken from food or feed. Biological material may be e.g. expectoration of any kind, broncheolavages, blood, skin tissue, biopsies, lymphocyte blood culture material, colonies, etc. Said samples may be prepared or extracted wording to any of the techniques known in the art.
The xe2x80x9ctargetxe2x80x9d trial in these samples may be either genomic DNA or precursor RNA of the organism to be detected (=target organism), or amplified versions thereof as set out above. More specifically, the nucleic acid sequence of the target material is localized in the spacer region of the target organism(s).
Detection and identification of the target material can be performed by using one of the many electrophoresis methods, hybridization methods or sequencing methods described in literature and currently known by men skilled in the art. However, a very convenient and advantageous technique for the simultaneous detection of nucleic acids possibly present in biological samples is the Line Probe Assay technique. The Line Probe Assay (LiPA) is a reverse hybridization format (Saiki et al., 1989) using membrane strips onto which several oligonucleotide probes (including negative or positive control oligonucleotides) can be conveniently applied as parallel lines.
The LiPA technique, as described by Stuyver et al. (1993) and in international application WO 94/12670, provides a very rapid and user-friendly hybridization test. Results can be read within 4 h, after the start of the amplification. After amplification during which usually a non-isotopic label is incorporated in the amplified product, and alkaline denaturation, the amplified product is contacted with the probes on the membrane and the hybridization is carried out for about 1 to 1,5 h. Consequently, the hybrids formed are detected by an enzymatic procedure resulting in a visual purple-brown precipitate. The LiPA format is completely compatible with commercially available scanning devices, thus rendering automatic interpretation of the results possible. All those advantages make the LiPA format liable for use in a routine setting.
The LiPA format is not only an advantageous tool for identification and detection of pathogens at the species level but also at higher or lower taxonomical levels. For instance, probe-configurations on LiPA strips can be selected in such a manner that they can detect a complete genus (e.g. Niesseria, Listera, etc.) or an identify subgroups within a genus (e.g. subgroups in the Mycobacterium avium-intracellulare-scrofulaceum complex) or can in some cases even detect subtypes (subspecies, serovars, sequevars, biovars, etc. whatever is clinically relevant) within a species.
It should be stressed that the ability to simultaneously generate hydridization results with a number of probes is an outstanding benefit of the LiPA technology. In many cases the amount of information which can be obtained by a particular combination of probes greatly outnumbers the data obtained by using single probe assays. Therefor the selection of probes an the membrane strip is of utmost importance since an optimized set of probes will generate the maximum of information possible. This is more particularly exemplified further herein.
The fact that different probes can be combined on one strip also offers the possibility to conveniently cope with a lack of sensitivity due to sequence heterogenity in the target region of the group of organisms to be detected. Due to this heterogenity, two or more probes may be required to positively identify all organisms of the particular group. These probes can be applied to membrane strips at different locations and the result is interpreted as positive if at least one of these probes is positive. Alternatively these probes can be applied as a mixture at the same location, hereby reducing the number of lines on a strip. This reduction may be convenient in order to make the strip more concise or to be able to extend the total number of probes on one strip. Another alternative approach, in view of its practical benefits, is the synthesis of oligonucleotides harbouring the sequences of two (or more) different probes (degenerate probes) which then can be further processed and applied to the strip as one oligonucleotide molecule. This approach would considerably simplify the manufacturing procedures of the LiPA-strips. For example, probes with nucleotide sequences A and B are both required to detect all strains of taxon X. In the latter alternative a probe can be synthesized having the nucleotide sequence AB. This probe will have the combined characteristics of probes A and B.
By virtue of the above-mentioned properties the LiPA system can be considered as a preferential format for a hybridization method wherein several organisms need to be detected simultaneously in a sample. Moreover, as described in the examples section, the LiPA system is a preferred format for a selection method for the experimental evaluation and selection of theoretically designed probes.
However, it should be clear that any other hybridization assay, whereby different probes are used under the same hybridization and wash conditions can be used for the above-mentioned detection and/or selection methods. For example, it may be possible to immobilize the target nucleic acid to a solid support, and use mixtures of different probes, all, differently labeled, resulting in a different detection signal for each of the probes hybridized to the target.
As an example, the procedure to be followed for the detection of one or more organisms in a sample using the LiPA format is outlined below:
First, the nucleic acids of the organism(s) to be detected in the sample, is made available for amplification and/or hybridization.
Secondly, the nucleic acids, if present, are amplified with one or another target amplification system, as specified below. Usually, amplification is need to enhance the subsequent hybridization signal. However for some samples or some organisms amplification might not be necessary. This might also be the case if, for the detection of the hybrids formed, highly sensitive signal-amplification systems are used.
Thirdly, eventually after a denaturation step, the nucleic acids present in the sample or the resulting amplified product are contacted with LiPA strips onto which one or more DNA-probes, allowing the detection of the organisms of interest, are immobilized, and hybridization is allowed to proceed.
Finally, eventually after having performed a wash step, the hybrids are detected using a convenient and compatible detection system. From the hybridization signals or patterns observed the presence or absence of one or several organisms screened for in that particular biological sample can be deduced.
The amplification system used may be more or less universal, depending on the specific application needed.
By using universal primers located in the conserved flanking regions (16S and 23S gene) or the rRNA spacer, the spacer region or most if not all organisms or eubacterial origin will be amplified. The same result may be obtained by using a combination of different sets of primers with reduced universality (multiplex amplification, i.e. an amplification procedure in which two or more primer sets are used simultaneously in one and the same reaction mixture).
For some applications it may be appropriate to amplify not all organisms present in the sample but more specifically, beforehand defined taxa. This may be achieved using specific primers located either in less conserved parts of the flanking genes of the spacers (e.g. MYCP1-5 for amplification of the spacer region of mycobacteria) or located in the spacers themselves (e.g. LIS-P1-P7, BRU-P1-4, CHTR-P1-2and YEC-P1-2 for specific amplification of the spacer regions) of Listeria species, Brucella species, Chlamydia trachomatis, and Yersinia enterocolitica respectively).
The present invention thus provides a method for detection and identification of at least one micro-organism, or for the simultaneous detection of several micro-organisms in a sample, comprising the steps of:
(i) if need be releasing, isolating and/or concentrating the polynucleic acids from the micro-organism(s) to be detected in the sample;
(ii) if need be amplifying the 16S-23S rRNA spacer region, or a part of it, from the micro-organism(s) to be detected, with at least one suitable primer pair;
(iii) hybridizing the polynucleic acids of step (i) or (ii) with a set of probes comprising at least two probes, under the same hybridization and wash conditions, with, said probes being selected from the sequences of table 1a or equivalents thereof and/or from taxon-specific probes derived from any of the spacer sequences represented in FIGS. 1-103, with said taxon-specific probe being selected such that it is capable of hybridizing under the same hybridization and wash conditions as at least one of the probes of table 1a;
(iv) detecting the hybrids formed in step (iii);
(v) identification of the micro-organism(s) present in the sample from the differential hybridization signals obtained in step (iv).
The probes as mentioned in table 1a are all selected in such a way that they show the desired hybridization characteristics at a hybridization and wash temperature of 50xc2x0 C. in a preferred hybridization and wash medium of 3xc3x97SSC and 20% formamide.
The term xe2x80x9cequivalentsxe2x80x9d of a probe, also called xe2x80x9cvariantsxe2x80x9d or xe2x80x9chomologuesxe2x80x9d or xe2x80x9cobvious derivativesxe2x80x9d, refers to probes differing in sequence from any of the probes specified in table 1 either by addition to or removal from any of their respective extremities of one or several nucleotides, or by changing one or more nucleotides within said sequences, or a combination of both, provided that said equivalents still hybridize with the same RNA or DNA target as the corresponding unmodified probe sequence. Said equivalents share at least 75% homology, preferably more than 80%, most preferably more than 85% homology with the corresponding unmodified probe sequence. It should be noted that, when using an equivalent of a probe, it may be necessary to modify the hybridization conditions to obtain the same specificity as the corresponding unmodified probe. As a consequence, since it is the aim of this invention to use a set of probes which work under the same hybridization and wash conditions, it will also be necessary to modify accordingly the sequence of the other probes, belonging to the same set as the original unmodified probe. These modifications can be done according to principles known in the art, e.g. such as those described in Hames B and Higgins S (Eds): Nucleic acid hybridization. Practical approach. IRL Press. Oxford, UK, 1985.
The invention also provides for a method to select taxon-specific probes from the spaces region sequence(s) of said taxon, said probes being selected such that they show their desired hybridization characteristics under unified hybridization and wash conditions.
The term xe2x80x9cunifiedxe2x80x9d conditions means that these conditions are the same for the different probes enabling the detection of different taxa.
Preferentially, the present invention provides for a method as described above wherein at least 2 micro-organisms are detected simultaneously.
In a preferred embodiment, the set of probes as described in step (iii) is comprising at least two probes being selected from the sequences of table 1a, or equivalents thereof.
In another embodiment, the set of probes as described in step (iii) is comprising at least one probe being selected from the sequences of table 1a, or equivalents thereof, and at least one taxon-specific probe derived from any of the spacer sequences as represented in FIGS. 1-103.
In still another embodiment, the set of probes as described in step (iii) is comprising at least two taxon-specific probes derived from any of the spacer sequences as represented in FIGS. 1-103.
The present invention also provides for a method as described above, wherein the probes as specified in step (iii) are combined with at least one other probe, preferentially also from the 16S-23S rRNA spar region, enabling the simultaneous detection of different pathogenic bacteria liable to be present in the same sample.
The organisms of clinical relevance present in biological samples may vary considerably depending on the origin of die sample. The most common pathogenic bacteria which may be found in sputum samples, or in samples originating from the respiratory tract, are:
Moraxella catarrhalis 
Streptococcus pneumomiae 
Haemophilus influenzae 
Pseudomonas aeruginosa 
Mycoplasma pneumomiae 
Acinetobacter species 
Mycobacterium species 
Staphylococcus aureus 
Legionella pneumophila 
A LiPA-strip harbouring spacer-probes enabling the detection of most if not all of these organisms would be extremely benificial for reasons explained above.
Evidently, this also applies for other biological samples, as there are: cerebrospinal fluid, urogenital samples, gastrointestinal samples, blood, urine, food products, soil, etc. For example, a preferred panel for cerebrospinal fluid would contain probe combinations enabling the detection and differentiation of the following organisms:
Neisseria meningitidis 
Streptococcus pneumoniae 
Streptococcus agalactiae 
Listeria monocytogenes 
Mycobacterium tuberculosis 
For some of the above mentioned organisms, spacer probes were already designed in a previous patent application (WO 91/16454). In order to be able to detect most pathogens possibly present in a sample in a single test, the probes of the present invention may be combined with at least one of the probes of WO 91/16454, or their obvious derivatives as specified in WO 91/16454. For clarity, these probes are listed hereafter:
Neisseria gonorrheoae: 
NGI1: CGATGCGTCGTTATTCTACTTCGC
NGI2: TTCGTTTACCTACCCGTTGACTAAGTAAGCAAAC
Neisseria meningitidis: 
NMI1: GGTCAAGTGTGACGTCGCCCTG
NMI2: GTTCTTGGTCAAGTGTGACGTC
NMI3: GCGTTCGTTATAGCTATCTACTGTGC
NMI4: TGCGTTCGATATTGCTATCTACTGTGCA
NMI5: TTTTGTTCTTGGTCAAGTGTGACGTCGCCCTGAATGGATTCTGTTCCATT
NMI6: TTTGCCTAACATTCCGTTGACTAGAACATCAGAC
Haemophilus ducrevi 
HDI1: TTATTATGCGCGAGCCATATTG
Branhamella catharralis 
BCI1: TTAAACATCTTACCAAAG
BCI2: TTGATGTTTAAACTTGCTTGGTGGA
Bordetella pertussis 
BPI1: CCACACCCATCCTCTGGACAGGCTT
Haemophilus influenzae 
HII1: ACGCATCAAATTGACCGCACTT
HII2: ACTTTGAAGTGAAAACTTAAAG
Streptococcus agalactiae 
SAI1: AATCGAAAGGTTCAAATTGTT
SAI2: GGAAACCTGCCATTTGCGTCTT
SAI3: TCCACGATCTAGAATTTAGATTGTAGAA
SAI4: TCTAGTTTTAAAGAAACTAGGTT
Streptococcus pneumoniae 
SPI1: GTGAGAGATCACCAAGTAATGCA
SPI2: AGGAACTGCGCATTGGTCTT
SPI3: GAGTTTATGACTGAAAGGTCAGAA
The invention thus provides for a method as described above, wherein said sample is originating from the respiratory tract, and wherein the set of probes as defined in step (iii) comprises at least one probe chosen from the following spacer probes:
MYC-ICG-1: ACTGGATAGTGGTTGCGAGCATCTA (SEQ ID NO 1)
MYC-ICG-22: CTTCTGAATAGTGGTTGCGAGCATCT (SEQ ID NO 2)
MTB-ICG-1: GGGTGCATGACAACAAAGTTGGCCA (SEQ ID NO 3)
MTB-ICG-2: GACTTGTTCCAGGTGTTGTCCCAC (SEQ ID NO 4)
MTB-ICG-3: CGGCTAGCGGTGGCGTGTTCT (SEQ ID NO 5)
MAI-ICG-1: CAACAGCAAATGATTGCCAGACACAC (SEQ ID NO 6)
MIL-ICG-11: GAGGGGTTCCCGTCTGTAGTG (SEQ ID NO 7)
MIL-ICG-22: TGAGGGGTTCTCGTCTGTAGTG (SEQ ID NO 8)
MAC-ICG-1: CACTCGGTCGATCCGTGTGGA (SEQ ID NO 9)
MAV-ICG-1: TCGGTCCGTCCGTGTGGAGTC (SEQ ID NO 10)
MAV-ICG-22: GTGGCCGGCGTTCATCGAAA (SEQ ID NO 11)
MIN-ICG-1: GCATAGTCCTTAGGGCTGATGCGTT (SEQ ID NO 12)
MIN-ICG-2: GCTGATGCGTTCGTCGAAATGTGTA (SEQ ID NO 13)
MIN-ICG-22: CTGATGCGTTCGTCGAAATGTGT (SEQ ID NO 14)
MEN-ICG-222: TGATGCGTTCGTCGAAATGTGT (SEQ ID NO 15)
MIN-ICG-2222: GGCTGATGCGTTCGTCGAAATGTGTAA (SEQ ID NO 16)
MAL-ICG-1: ACTAGATGAACGCGTAGTCCTTGT (SEQ ID NO 17)
MHEF-ICG-1: TGGACGAAAACCGGGTGCACAA (SEQ ID NO 18)
MAH-ICG-1: GTGTAATTTCTTTTTTAACTCTTGTGTGTAAGTAAGTG (SEQ ID NO 19)
MCO-ICG-11: TGGCCGGCGTGTTCATCGAAA (SEQ ID NO 20)
MTH-ICG-11: GCACTTCAATTGGTGAAGTGCGAGCC (SEQ ID NO 21)
MTH-ICG-2: GCGTGGTCTTCATGGCCGG (SEQ ID NO 22)
MEF-ICG-11: ACGCGTGGTCCTTCGTGG (SEQ ID NO 23)
MSC-ICG-1: TCGGCTCGTTCTGAGTGGTGTC (SEQ ID NO 24)
MKA-ICG-1: GATGCGTTTGCTACGGGTAGCGT (SEQ ID NO 25)
MKA-ICG-2: GATGCGTTGCCTACGGGTAGCGT (SEQ ID NO 26)
MKA-ICG-3: ATGCGTTGCCCTACGGGTAGCGT (SEQ ID NO 27)
MKA-ICG-4: CGGGCTCTGTTCGAGAGTTGTC (SEQ ID NO 28)
MKA-ICG-5: CCCTCAGGGATTTTCTGGGTGTTG (SEQ ID NO 182)
MKA-ICG-6: GGACTCGTCCAAGAGTGTTGTCC (SEQ ID NO 183)
MKA-ICG-7: TCGGGCTTGGCCAGAGCTGTT (SEQ ID NO 184)
MKA-ICG-8: GGGTGCGCAACAGCAAGCGA (SEQ ID NO 185)
MKA-ICG-9: GATGCGTTGCCCCTACGGG (SEQ ID NO 186)
MKA-ICG-10: CCCTACGGGTAGCGTGTTCTTTTG (SEQ ID NO 187)
MCH-ICG-1: GGTGTGGACTTTGACTTCTGAATAG (SEQ ID NO 29)
MCH-ICG-2: CGGCAAAACGTCGGACTGTCA (SEQ ID NO 30)
MCH-ICG-3: GGTGTGGTCCTTGACTTATGGATAG (SEQ ID NO 210)
MGO-ICG-1: AACACCCTCGGGTGCTGTCC (SEQ ID NO 31)
MCO-ICG-2: GTATGCGTTGTCGTTCGCGGC (SEQ ID NO 32)
MGO-ICG-5: CGTGAGGGGTCATCGTCTGTAG (SEQ ID NO 33)
MUL-ICG-1: GGTTTCGGGATGTTGTCCCACC (SEQ ID NO 175)
MGV-ICG-1: CGACTGAGGTCGACGTGGTGT (SEQ ID NO 176)
MGV-ICG-2: GGTGTTTGAGCATTGAATAGTGGTTGC (SEQ ID NO 177)
MGV-ICG-3: TCGGGCCGCGTGTTCGTCAAA (SEQ ID NO 211)
MXE-ICG-1: GTTGGGCAGCAGGCAGTAACC (SEQ ID NO 178)
MSI-ICG-1: CCGGCAACGGTTACGTGTTC (SEQ ID NO 179)
MFO-ICG-1: TCGTTGGATGGCCTCGCACCT (SEQ ID NO 180)
MFO-ICG-2: ACTTGGCGTGGGATGCGGGAA (SEQ ID NO 181)
MML-ICG-1: CGGATCCATTGAGTCCTTGTCCC (SEQ ID NO 188)
MML-ICG-2: TCTAAATGAACGCACTGCCGATGG (SEQ ID NO 189)
MCE-ICG-1: TGAGGGAGCCCGTGCCTGTA (SEQ ID NO 190)
MHP-ICG-1: CATGTTGGGCTTGATCGGGTGC (SEQ ID NO 191)
PA-ICG 1: TGGTGTGCTGCGTGATCCGAT (SEQ ID NO 34)
PA-ICG 2: TGAATGTTCGTGGATGAACATTGATT (SEQ ID NO 35)
PA-ICG 3: CACTGGTGATCATTCAACTCAAG (SEQ ID NO 36)
PA-ICG 4: TGAATGTTCGT(G/A)(G/A)ATGAACATTGATTTCTGGTC (SEQ ID NO 37)
PA-ICG 5: CTCTTTCACTGGTGATCATTCAAGTCAAG (SEQ ID NO 38)
MPN-ICG 1: ATCGGTGGTAAATTAAACCCAAATCCCTGT (SEQ ID NO 49)
MPN-ICG 2: CAGTTCTGAAAGAACATTTCCGCTTCTTTC (SEQ ID NO 50)
MGE-ICG 1: CACCCATTAATTTTTTCGGTGTTAAAACCC (SEQ ID NO 51)
Mycoplasma-ICG: CAAAACTGAAAACGACAATCTTTCTAGTTCC (SEQ ID NO 52)
STAU-ICG 1: TACCAAGCAAAACCGAGTGAATAAAGAGTT (SEQ ID NO 53)
STAU-ICG 2: CAGAAGATGCGGAATAACGTGAC (SEQ ID NO 54)
STAU-ICG 3: AACGAAGCCGTATGTGAGCATTTGAC (SEQ ID NO 55)
STAU-ICG 4: GAACGTAACTTCATGTTAACGTTTGACTTAT (SEQ ID NO 56)
ACI-ICG 1: GCTTAAGTGCACAGTGCTCTAAACTGA (SEQ ID NO 57)
ACI-ICG 2: CACGGTAATTAGTGTGATCTGACGAAG (SEQ ID NO 58)
and more preferably from the following spacer probes:
MYC-ICG-1: ACTGGATAGTGGTTGCGAGCATCTA (SEQ ID NO 1)
MYC-ICG-22: CTTCTGAATAGTGTTGCGAGCATCT (SEQ ID NO 2)
MTB-ICG-1: GGGTGCATGACAACAAAGTTGGCCA (SEQ ID NO 3)
MTB-ICG-2: GACTTGTTCCAGGTGTTGTCCCAC (SEQ ID NO 4)
MTB-ICG-3: CGGCTACCGGTGGCGTGTTCT (SEQ ID NO 5)
MAI-ICG-1: CAACAGCAAATGATTGCCAGACACAC (SEQ ID NO 6)
MIL-ICG-11: GAGGGGTTCCCGTCTGTAGTG (SEQ ID NO 7)
MIL-ICG-22: TGAGGGGTTCTCGTCTGTAGTG (SEQ ID NO 8)
MAC-ICG-1: CACTCGGTCGATCCGTGTGGA (SEQ ID NO 9)
MAV-ICG-1: TCGGTCCGTCCGTGTGGAGTC (SEQ ID NO 10)
MAV-ICG-22: GTGGCCGGCGTTCATCGAAA (SEQ ID NO 11)
MIN-ICG-1: GCATAGTCCTTAGGCCTGATGCGTT (SEQ ID NO 12)
MAL-ICG-1: ACTAGATGAACGCGTAGTCCTTGT (SEQ ID NO 17)
MCO-ICG-11: TGGCCGGCGTGTTCATCGAAA (SEQ ID NO 20)
MTH-ICG-11: GCACTTCAATTGGTGAAGTGCGAGCC (SEQ ID NO 21)
MTH-ICG-2: GCGTGCTCTTCATGGCCGG (SEQ ID NO 22)
MEF-ICG-11: ACGCGTGGTCCTTCGTGG (SEQ ID NO 23)
MSC-ICG-1: TCGGCTCGTTCTGAGTGGTGTC (SEQ ID NO 24)
MKA-ICG-3: ATGCGTTGCCCTACGGGTAGCGT (SEQ ID NO 27)
MKA-ICG-4: CGGGCTCTGTTCGAGAGTTGTC (SEQ ID NO 28)
MKA-ICG-5: CCCTCAGGGATTTTCTGGGTGTTG (SEQ ID NO 182)
MKA-ICG-6: GGACTCGTCCAAGAGTGTTGTCC (SEQ ID NO 183)
MKA-ICG-7: TCGGGCTTGGCCAGAGCTGTT (SEQ ID NO 184)
MKA-ICG-8: GGGTGCGCAACAGCAAGCGA (SEQ ID NO 185)
MKA-ICG-9: GATGCGTTGCCCCTACGGG (SEQ ID NO 186)
MKA-ICG-10: CCCTACGGGTAGCGTGTTCTTTTG (SEQ ID NO 187)
MCH-ICG-1: GGTGTGGACTTTGACTTCTGAATAG (SEQ ID NO 29)
MCH-ICG-2: CGGCAAAACGTCGGACTGTCA (SEQ ID NO 30)
MCH-ICG-3: GGTGTGGTCCTTGACTTATGGATAG (SEQ ID NO 210)
MGO-ICG-5: CGTGAGGGGTCATCGTCTGTAG (SEQ ID NO 33)
MUL-ICG-1: GGTTTCGGGATGTTGTCCCACC (SEQ ID NO 175)
MGV-ICG-1: CGACTGAGGTCGACGTGGTGT (SEQ ID NO 176)
MGV-ICG-2: GGTGTTTGAGCATTGAATAGTGGTTGC (SEQ ID NO 177)
MGV-ICG-3: TCGGGCCGCGTGTTCGTCAAA (SEQ ID NO 211)
MXE-ICG-1: GTTGGGCACCAGGCAGTAACC (SEQ ID NO 178)
MSI-ICG-1: CCGGCAACGGTTACGTGTTC (SEQ ID NO 179)
MFO-ICG-1: TCGTTGGATGGCCTCGCACCT (SEQ ID NO 180)
MFO-ICG-2: ACTTGGCGTGGGATGCGGGAA (SEQ ID NO 181)
MML-ICG-1: CGGATCGATTGAGTGCTTGTCCC (SEQ ID NO 188)
MML-ICG-2: TCTAAATGAACGCACTGCCGATGG (SEQ ID NO 189)
MCE-ICG-1: TGAGGGAGCCCGTGCCTGTA (SEQ ID NO 190)
MHP-ICG-1: CATGTTGGGCTTGATCGGGTGC (SEQ ID NO 191)
PA-ICG 1: TGGTGTGCTGCGTGATCCGAT (SEQ ID NO 34)
PA-ICG 4: TGAATGTTCGT(G/A)(G/A)ATGAACATTGATTTCTGGTC (SEQ ID NO 37)
PA-ICG 5: CTCTTTCACTGGTGATCATTCAAGTCAAG (SEQ ID NO 38)
MPN-ICG 1: ATCGGTGGTAAATTAAACCCAAATCCCTGT (SEQ ID NO 49)
MPN-ICG 2: CAGTTCTGAAAGAACATTTCCGCTTCTTTC (SEQ ID NO 50)
MGE-ICG 1: CACCCATTAATTTTTTCGGTGTTAAAACCC (SEQ ID NO 51)
Mycoplasma-ICG: CAAAACTGAAAACGACAATCTTTCTAGTTCC (SEQ ID NO 52)
STAU-ICG 1: TACCAAGCAAAACCGAGTGAATAAAGAGTT (SEQ ID NO 53)
STAU-ICG 2: CAGAAGATGCGGAATAACGTGAC (SEQ ID NO 54)
STAU-ICG 3: AACGAAGCCGTATGTGAGCATTTGAC (SEQ ID NO 55)
STAU-ICG 4: GAACGTAACTTCATGTTAACGTTTGACTTAT (SEQ ID NO 56)
ACI-ICG 1: GCTTAAGTGCACAGTGCTCTAAACTGA (SEQ ID NO 57)
ACI-ICG 2: CACGGTAATTAGTGTGATCTGACGAAG (SEQ ID NO 58)
or equivalents of said probes,
and/or wherein the set of probes comprises at least one taxon-specific probe derived from the spacer region sequence corresponding to one of the micro-organisms to be detected in said sample, said spacer region sequence being chosen from any of the sequences as represented by SEQ ID NO 76 to 106, 157 to 174, 124, 125, 111 to 115, 139 to 144, or 126 to 130, and with said probes or equivalents being possibly used in combination with any probe detecting at least one of the following organisms: Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis or Bordetella pertussis. 
The above mentioned probes of the invention are designed for the detection of Mycobacterium species (SEQ ID NO 1 to 33 and 175 to 191), of Pseudomonas aeruginosa (SEQ ID NO 34 to 38), of Mycoplasma species (SEQ ID NO 49 to 52), of Staphylococcus aureus (SEQ ID NO 53 to 56) and of Acinetobacter baumanii (SEQ ID NO 57 and 58).
Preferentially, at least two, three, four, five, six, seven, eight or more of said probes are used simultaneously.
The invention also relates to a method as described above, wherein said sample is a sample taken from the cerebrospinal fluid, and wherein the set of probes as described in step (iii) comprises at least one probe chosen from the following spacer probes:
MYC-ICG-1: ACTGGATAGTGGTTGCGAGCATCTA (SEQ ID NO 1)
MYC-ICG-22: CTTCTGAATAGTGGTTGCGAGCATCT (SEQ ID NO 2)
MTB-ICG-1: GGGTGCATGACAACAAAGTTGGCCA (SEQ ID NO 3)
MTB-ICG-2: GACTTGTTCCAGGTGTTGTCCCAC (SEQ ID 4)
MTB-ICG-3: CGGCTAGCGGTGGCGTGTTCT (SEQ ID NO 5)
LIS-ICG 1: CAAGTAACCGAGAATCATCTGAAAGTGAATC (SEQ ID NO 39)
LMO-ICG 1: TTTTCAACCTTTACTTCGTAGAAGTAAATTGGTTAAG (SEQ ID NO 40)
LMO-ICG 2: TGAGAGGTTAGTACTTCTCAGTATGTTTGTTC (SEQ ID NO 41)
LMO-ICG 3: AGGCACTATGCTTGAAGCATCGC (SEQ ID NO 42)
LISP-ICG 1: CGTTTTCATAAGCGATCGCACGTT (SEQ ID NO 212)
and preferably from the following spacer probes:
MYC-ICG-1: ACTGGATAGTGGTTGCGAGCATCTA (SEQ ID NO 1)
MYC-ICG-22: CTTCTGAATAGTGGTTGCGAGCATCT (SEQ ID NO 2)
MTB-ICG-1: GGGTGCATGACAACAAAGTTGGCCA (SEQ ID NO 3)
MTB-ICG-2: GACTTGTCCAGGTGTTGTCCCAC (SEQ ID NO 4)
MTB-ICG-3: CGGCTAGCGGTGGCGTGTTCT (SEQ ID NO 5)
LIS-ICG 1: CAAGTAACCGAGAATCATCTGAAAGTGAATC (SEQ ID NO 39)
LMO-ICG 3: AGGCACTATGCTTGAAGCATCGC (SEQ ID NO 42)
LISP-ICG 1: CGTTTTCATAAGCGATCGCACGTT (SEQ ID NO 212)
or equivalents of said probes,
and/or wherein the set of probes comprises at least one taxon-specific probe derived from the spacer region sequence corresponding to one of the micro-organisms to be detected in said sample, said spacer region sequence being chosen from any of the sequences as represented by SEQ ID NO 116, 118-121, or 213-215,
and with said probes or equivalents being possibly used in combination with any probe detecting at least one of the following organisms: Neisseria meningitidis, Haemophilus influenzae or Streptococcus pneumoniae. 
The above mentioned probes of the invention are designed for the detection of Mycobacterium species, and more particularly Mycobacterium tuberculosis (SEQ ID NO 1 to 5), and of Listeria species; more particularly Listeria monocytogenes (SEQ ID NO 39 to 42).
Preferentially, at least two, three, four, five, six, seven, eight or more of said probes are used simultaneously.
The invention also relates to a method as described above, wherein said sample is a sample taken from the urogenital tract, and wherein the set of probes as described in step (iii) comprises at least one probe chosen from the following spacer probes:
CHTR-ICG 1: GGAAGAAGCCTGAGAAGGTTTCTGAC (SEQ ID NO 45)
CHTR-ICG 2: GCATTTATATGTAAGACCAAGCATTCTATTTCA (SEQ ID NO 46)
CHTR-ICG 3: GAGTAGCGTGGTGAGGACGAGA (SEQ ID NO 47)
CHTR-ICG 4: GAGTAGCGCGGTGAGGACGAGA (SEQ ID NO 201)
CHPS-ICG 1: GGATAACTCTCTTAGGACGGTTTGAC (SEQ ID NO 48)
MGE-ICG 1: CACCCATTAATTTTTTCGGTGTTAAAACCC (SEQ ID NO 51)
Mycoplasma-ICG: CAAAACTOAAAACGACAATCTTTCTAGTTCC (SEQ ID NO 52)
or equivalents of said probes,
and/or wherein the set of probes comprises at least one taxon-specific probe derived from the spacer region sequence corresponding to one of the micro-organisms to be detected in said sample, said spacer region sequence being chosen from any of the sequences as represented by SEQ ID NO 122, 123, 197, 124 or 125,
with said probes or equivalents being possibly used in combination with any probe detecting at least one of the following organisms: Neisseria gonorrhoeae, Haemophilus ducrevi or Streptococcus agalactiae. 
The above mentioned probes of the invention are designed for the detection of Chlamydia species (SEQ ID NO 45 to 48 and 201) and of Mycoplasma species (SEQ ID NO 51 and 52).
Preferentially, at least two, three, four, five, six or seven of said probes are used simultaneously.
The invention also relates to a method as described above, wherein said sample is a sample taken from food, and wherein the set of probes as defined in step (iii) comprises at least one probe chosen from the following spar probes:
LIS-ICG 1: CTTGTAACCGAGAATCATCTGAAAGTGAATC (SEQ ID NO 39)
LMO-ICG 1: AAACAACCTTTACTTCGTAGAAGTAAATTGGTTAAG (SEQ ID NO 40)
LMO-ICG 2: TGAGAGGTTAGTACTTCTCAGTATGTTTGTTC (SEQ ID NO 41)
LMO-ICG 3: AGGCACTATGCTTGAAGCATCGC (SEQ ID NO 42)
LIV-ICG 1: GITAGCATAAATAGGTAACTATTTATGACACAAGTAAC (SEQ ID NO 43)
LSE-ICG 1: AGTTAGCATAAGTAGTGTAACTATTTATGACACAAG (SEQ ID NO 44)
LISP-ICG 1: CGTTTTCATAAGCGATCGCACGTT (SEQ ID NO 212)
STAU-ICG 1: TACCAAGCAAAACCGAGTGAATAAAGAGTT (SEQ ID NO 53)
STAU-ICG 2: CAGAAGATGCGGAATAACGTGAC (SEQ ID NO 54)
STAU-ICG 3: AACGAAGCCGTATGTGAGCATTTGAC (SEQ ID NO 55)
STAU-ICG 4: GAACGTAACTTCATGTTAACGTTTGACTTAT (SEQ ID NO 56)
BRU-ICG 1: CGTGCCGCCTTCGTTTCTCTTT (SEQ ID NO 59)
BRU-ICG 2: TTCGCTTCGGGGTGGATCTGTG (SEQ ID NO 60)
BRU-ICG 3: GCGTAGTAGCGTTTGCGTCGG (SEQ ID NO 193)
BRU-ICG 4: CCCAAGAAGCTTGCTCAAGCC (SEQ ID NO 194)
SALM-ICG 1: CAAAACTGACTTACGAGTCACGTTTGAG (SEQ ID NO 61)
SALM-ICG 2: GATGTATGCTTCGTTATTCCACGCC (SEQ ID NO 62)
STY-ICG 1: GGTCAAACCTCCAGGGACGCC (SEQ ID NO 63)
SED-ICG 1: GCGGTAATGTGTGAAAGCGTTGCC (SEQ ID NO 64)
YEC-ICG 1: GGAAAAGGTACTGCACGTGACTG (SEQ ID NO 198)
YEC-ICG 2: GACAGCTGAAACTTATCCCTCCG (SEQ ID NO 199)
YEC-ICG 3: GCTACCTGTTGATGTAATGAGTCAC (SEQ ID NO 200)
and preferably from the following spacer probes:
LIS-ICG 1: CAAGTAACCGAGAATCATCTGAAAGTGAATC (SEQ ID NO 39)
LMO-ICG 3: AGGCACTATGCTTGAAGCATCGC (SEQ ID NO 42)
LISP-ICG 1: CGTTTTCATAAGCGATCGCACGTT (SEQ ID NO 212)
STAU-ICG 1: TACCAAGCAAAACCGAGTGAATAAAGAGTT (SEQ ID NO 53)
STAU-ICG 2: CAGAAGATGCGGAATTTCGTGAC (SEQ ID NO 54)
STAU-ICG 3: AACGAAGCCGTATGTGAGCATTTGAC (SEQ ID NO 55)
STAU-ICG 4: GAACGTAACTTCATGTTAACGTTTGACTTAT (SEQ ID NO 56)
BRU-ICG 2: TTCGCTTCGGGGTGGATCTGTG (SEQ ID NO 60)
BRU-ICG 3: GCGTAGTAGCGTTTGCGTCGG (SEQ ID NO 193)
BRU-ICG 4: CGCAAGAAGCTTGCTCAAGCC (SEQ ID NO 194)
SALM-ICG 1: CAAAACTGACTTACGAGTCACGTTTGAG (SEQ ID NO 61)
YEC-ICG 1: GGAAAAGGTACTGCCACGTGACTG (SEQ ID NO 198)
YEC-ICG 2: GACAGCTGAAACTTATCCCTCCG (SEQ ID NO 199)
YEC-ICG 3: GCTACCTGTTGATGTTATGAGTCAC (SEQ ID NO 200)
or equivalents of said probes,
and/or wherein the set of probes comprises at least one taxon-specific probe derived from the spacer region sequence corresponding to one of the micro-organisms to be detected in said sample, said spacer region sequence being chosen from any of the sequences as represented by SEQ ID NO 116, 118-121, 213-215, 139-144, 131, 132, 154, 133-138, 195 or 196,
with said probes or equivalents being possibly used in combination with any probe detecting strains of Campylobacter species.
The above mentioned probes of the invention are designed for the detection of Listeria species (SEQ ID NO 39 to 44), of Staphylococcus species (SEQ ID NO 53 to 56), of Brucella species (SEQ ID NO 59, 60, 193 and 194), of Salmonella species (SEQ ID NO 61 to 64) and of Yersinia enterocolitica (SEQ ID NO 198 to 200).
Preferentially, at least two, three, four, five, six, seven, eight or more of said probes are used simultaneously.
The invention also relates to a method as described above, wherein said sample is a sample taken from the gastrointestinal tract of a patient, and wherein the set of probes as defined in step (iii) comprises at least one probe chosen from the following spacer probes:
SALM-ICG 1: CAAAACTGACTTACGAGTCACGTTTGAG (SEQ ID NO 61)
SALM-ICG 2: GATGTATGCTTCGTTATTCCACGCC (SEQ ID NO 62)
STY-ICG 1: GGTCAAACCTCCAGGGACGCC (SEQ ID NO 63)
SED-ICG 1: GCGCTAATGTCTGAAAGCGTTGCC (SEQ ID NO 64)
YEC-ICG 1: GGAAAAGGTACTGCACGTGACTG (SEQ ID NO 198)
YEC-ICG 2: GACAGCTGAAACTTATCCCTCCG (SEQ ID NO 199)
YEC-ICG 3: GCTACCTGTTGATGTAATGAGTCAC (SEQ ID NO 200)
and preferably from the following spacer probes:
SALM-ICG 1: CAAAACTGACTTACGAGTCACGTTTGAG (SEQ ID NO 61)
YEC-ICG 1: GGAAAAGGTACTGCACGTGACTG (SEQ ID NO 198)
YEC-ICG 2: GACAGCTGAAACTTATCCCTCCG (SEQ ID NO 199)
YEC-ICG 3: GCTACCTGTTGATGTAATGAGTCAC (SEQ ID NO 200)
or equivalents of said probes,
and/or wherein the set of probes comprises at least one taxon-specific probe derived from the spacer region sequence corresponding to one of the micro-organisms to be detected in said sample, said spacer region sequence being chosen from any of the sequences as represented by SEQ ID NO 133-138 or 195-196,
with said probes or equivalent being possibly used in combination with any probe detecting Campylobacter species.
The above mentioned probes of the invention are designed to detect Salmonella species (SEQ ID NO 61 to 64) and Yersinia enterocolitica (SEQ ID NO 198 to 200).
Preferentially, at least two, three, four, five, six or seven of said probes are used simultaneously.
The invention also relates to the use of the selected probes or their equivalents for the detection of specific bacterial taxa, said taxa being either a complete genus, or a subgroup within a genus, a species, or even a subtype within a species.
The invention thus provides for a method as described above to detect and identify one or more strains of Mycobacterium species and subspecies in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MYC-ICG-1: ACTGGATAGTGGTTGCGAGCATCTA (SEQ ID NO 1)
MYC-ICG-22: CTTCTGAATAGTGGTTGCGAGCATCT (SEQ ID NO 2)
MTB-ICG-1: GGGTGCATGACAACAAAGTTGGCCA (SEQ ID NO 3)
MTB-ICG-2: GACTTGTTCCAGGTGTTGTCCCAC (SEQ ID NO 4)
MTB-ICG-3: CGGCTAGCGGTGGCGTGTTCT (SEQ ID NO 5)
MAI-ICG-1: CAACAGCAAATGATTGCCAGACACAC (SEQ ID NO 6)
MIL-ICG-11: GAGGGGTTCCCGTCTGTAGTG (SEQ ID NO 7)
MIL-ICG-22: TGAGGGGTTCTCGTCTGTAGTG (SEQ ID NO 8)
MAC-ICG-1: CACTCGGTCGATCCGTGTGGA (SEQ ID NO 9)
MAV-ICG-1: TCGGTCCGTCCGTGTGGAGTC (SEQ ID NO 10)
MAV-ICG-22: GTGGCCGGCGTTCATCGAAA (SEQ ID NO 11)
KIN-ICG-1: GCATAGTCCTTAGGGCTGATGCGTT (SEQ ID NO 12)
MIN-ICG-2: GCTGATGCGTTCGTCGAAATGTGTA (SEQ ID NO 13)
MIN-ICG-22: CTGATGCGTTCGTCGAAATGTGT (SEQ ID NO 14)
MIN-ICG-22: TGATGCGTTCGTCGAAATGTGT (SEQ ID NO 15)
MIN-ICG-2222: GGCTGATGCGTTCGTCGAAATGTGTAA (SEQ ID NO 16)
MAL-ICG-1: ACTAGATGAACGCGTAGTCCTTGT (SEQ ID NO 17)
MHEF-ICG-1: TGGACGAAAACCGGGTGCACAA (SEQ ID NO 18)
MAH-ICG-1: GTGTAATTTCTTTTTTAACTCTTGTGTGTAAGTAAGTG (SEQ ID NO 19)
MCO-ICG-11: TGGCCGGCGTGTTCATCGAAA (SEQ ID NO 20)
MTH-ICG-11: GCACTTCAATTGGTGAAGTGCGAGCC (SEQ ID NO 21)
MTH-ICG-2: GCGTGGTCTTCATGGCCGG (SEQ ID NO 22)
MEF-ICG-11: ACGCGTGGTCCTTCGTGG (SEQ ID NO 23)
MSC-ICG-1: TCGGCTCGTTCTGAGTGGTGTC (SEQ ID NO 24)
MKA-ICG-1: GATGCGTTTGCTACGGGTAGCGT (SEQ ID NO 25)
MKA-ICG-2: GATGCGTTGCCTACGGGTAGCGT (SEQ ID NO 26)
MKA-ICG-3: ATGCGTTGCCCTACGGGTAGCGT (SEQ ID NO 27)
MKA-ICG-4: CGGGCTCTGTTCGAGAGTTGTC (SEQ ID NO 28)
MKA-ICG-5: CCCTCAGGGATTTTCTGGGTGTTG (SEQ ID NO 182)
MKA-ICG-6: GGACTCGTCCAAGAGTGTTGTCC (SEQ ID NO 183)
MKA-ICG-7: TCGGGCTTGGCCAGAGCTGTT (SEQ ID NO 184)
MKA-ICG-8: GGGTGCGCAACAGCAAGCGA (SEQ ID NO 185)
MKA-ICG-9: GATGCGTTGCCCCTACGGG (SEQ ID NO 186)
MKA-ICG-10: CCCTACGGGTAGCGTGTTCTTTTG (SEQ ID NO 187)
MCH-ICG-1: GGTGTGGACTTTGACTTCTGAATAG (SEQ ID NO 29)
MCH-ICG-2: CGGCAAAACGTCGGACTGTCA (SEQ ID NO 30)
MGO-ICG-1: AACACCCTCGGGTGCTGTCC (SEQ ID NO 31)
MCO-ICG-2: GTATGCGTTGTCGTTCGCGGC (SEQ ID NO 32)
MGO-ICG-5: CGTGAGGGGTCATCGTCTGTAG (SEQ ID NO 33)
MUL-ICG-1: GGTTTCGGGATGTTGTCCCACC (SEQ ID NO 175)
MGV-ICG-1: CGACTGAGGTCGACGTGGTGT (SEQ ID NO 176)
MGV-ICG-2: GGTGTTTGAGCATTGAATAGTGGTTGC (SEQ ID NO 177)
MXE-ICG-1: GTTGGGCAGCAGGCAGTAACC (SEQ ID NO 178)
MSI-ICG-1: CCGGCAACGGTTACGTGTTC (SEQ ID NO 179)
MFO-ICG-1: TCGTTGGATGGCCTCGCACCT (SEQ ID NO 180)
MFO-ICG-2: ACTTGGCGTGGGATGCGGGAA (SEQ ID NO 181)
MML-ICG-1: CGGATCGATTGAGTGCTTGTCCC (SEQ ID NO 188)
MML-ICG-2: TCTAAATGAACGCACTGCCGATGG (SEQ ID NO 189)
MCE-ICG-1: TGAGGGAGCCCGTGCCTGTA (SEQ ID NO 190)
MHP-ICG-1: CATGTTGGGCTTGATCGGGTGC (SEQ ID NO 191)
and more preferably to at least one probe of the following restricted group of spacer probes:
MYC-ICG-1: ACTGGATAGTGGTTGCGAGCATCTA (SEQ ID NO 1)
MYC-ICG-22: CTTCTGAATAGTGGTTGCGAGCATCT (SEQ ID NO 2)
MTB-ICG-1: GGGTGCATGACAACAAAGTTGGCCA (SEQ ID NO 3)
MTB-ICG-2: GACTTGTTCCAGGTGTTGTCCCAC (SEQ ID NO 4)
MTB-ICG-3: CGGCTAGCGGTGGCGTGTTCT (SEQ ID NO 5)
MAI-ICG-1: CAACAGCAAATGATTGCCAGACACAC (SEQ ID NO 6)
MIL-ICG-11: GAGGGGTTCCCGTCTGTAGTG (SEQ ID NO 7)
MIL-ICG-22: TGAGGGGTCTCGTCTGTAGTG (SEQ ID NO 8)
MAC-ICG-1: CACTCGGTCGATCCGTGTGGA (SEQ ID NO 9)
MAV-ICG-1: TCGGTCCGTCCGTGTGGAGTC (SEQ ID NO 10)
MAV-ICG-22: GTGGCCGGCGTTCATCGAAA (SEQ ID NO 11)
MIN-ICG-1: GCATAGTCCTTAGGGCTGATGCGTT (SEQ ID NO 12)
MAL-ICG-1: ACtAGATGAACGCGTAGTCCITGT (SEQ ID NO 17)
MCO-ICG-11: TGGCCGGCGTGTTCATCGAAA (SEQ ID NO 20)
MTH-ICG-11: GCACTTCAATTGGTGAAGTGCGAGCC (SEQ ID NO 21)
MTH-ICG-2: GCGTGGTCTTCATGGCCGG (SEQ ID NO 22)
MEF-ICG-11: ACGCGTGGTCCTTCGTGG (SEQ ID NO 23)
MSC-ICG-1: TCGGCTCGTTCTGAGTGGTGTC (SEQ ID NO 24)
MKA-ICG-3: ATGCGTTGCCCTACGGGTAGCGT (SEQ ID NO 27)
MKA-ICG-4: CGGGCTCTGTTCGAGAGTTGTC (SEQ ID NO 28)
MKA-ICG-5: CCCTCAGGGATTTTCTGGGTGTTG (SEQ ID NO 182)
MKA-ICG-6: GGACTCGTCCAAGAGTGTTGTCC (SEQ ID NO 183)
MKA-ICG-7: TCGGGCTTGGCCAGAGCTGTT (SEQ ID NO 184)
MKA-ICG-8: GGGTGCGCAACAGCAAGCGA (SEQ ID NO 185)
MKA-ICG-9: GATGCGTTGCCCCTACGGG (SEQ ID NO 186)
MKA-ICG-10: CCCTACGGGTAGCGTGTTCTTTTG (SEQ ID NO 187)
MCH-ICG-1: GGTGGACTTTGACTTCTGAATAG (SEQ ID NO 29)
MCH-ICG-2: CGGCAAAACGTCGGACTGTCA (SEQ ID NO 30)
MCH-ICG-3: GGTGTGGTCCTTGACTTATGGATAG (SEQ ID NO 210)
MGO-ICG-5: CGTGAGGGGTCATCGTCTGTAG (SEQ ID NO 33)
MUL-ICG-1: GGTTTCGGGATGTTGTCCCACC (SEQ ID NO 175)
MGV-ICG-1: CGACTGAGGTCGACGTGGTGT (SEQ ID NO 176)
MGV-ICG-2: GGTGTTTGAGCATTGAATAGTGGTTGC (SEQ ID NO 177)
MGV-ICG-3: TCGGGCCGCGTGTTCGTCAAA (SEQ ID NO 211)
MXE-ICG-1: GTCGGGCAGCAGGCAGTAACC (SEQ ID NO 178)
MSI-ICG-1: CCGCCAACGGTTACGTGTTC (SEQ ID NO 179)
MFO-ICG-1: TCGTTGGATGGCCTCGCACCT (SEQ ID NO 180)
MFO-ICG-2: ACTTGGCGTGGGATGCGGGAA (SEQ ID NO 181)
MML-ICG-1: CGGATCGATTGAGTGCTTGTCCC (SEQ ID NO 188)
MML-ICG-2: TCTAAATGAACGCACTGCCGATGG (SEQ ID NO 189)
MCE-ICG-1: TGAGGGAGCCCGTGCCTGTA (SEQ ID NO 190)
MHP-ICG-1: CATGTTGGGCTTGATCGGGTGC (SEQ ID NO 191)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 76-110, or 157-174 provided said probe hybridizes specifically to a Mycobacterium species.
The sequences represented by SEQ ID NO 76-110 and 157-174 are new.
Preferentially, at least two, three, four, five, six, seven, eight or more of said probes are used simultaneously.
As described above, the preferred restricted set of probes are those probes which showed a sensitivity and specificity of more than 80%, preferably more than 90%, most preferably more than 95%, under the specific hybridization conditions as described in the examples section.
In one specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium tuberculosis complex strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MTB-ICG-1: GGGTGCATGACAACAAAGTTGGCCA (SEQ ID NO 3)
MTB-ICG-2: GACTTGTTCCAGGTGTTGTCCCAC (SEQ ID NO 4)
MTB-ICG-3: CGGCTAGCGGTGGCGTGTTCT (SEQ ID NO 5)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 76 provided said probe hybridizes specifically to the M. tuberculosis complex. The M. tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis BCG, M. africanum and M. microri strains.
The sequence represented in SEQ ID NO 76 is new.
Preferentially, at least two, or three of said probes are used simultaneously.
In another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium strains from the MAIS-complex, wherein step (iii) comprises hybridizing to at least one of the following probes:
MAI-ICG-1: CAACACCAAATGATTGCCAGACACAC (SEQ ID NO 6)
MIL-ICG-11: GAGGGGTFCCCGTCTGTAGTG (SEQ ID NO 7)
MIL-ICG-22: TGAGGGGTTCTCGTCTGTAGTG (SEQ ID NO 8)
MAC-ICG-1: CACTCGGTCGATCCGTGTGGA (SEQ ID NO 9)
MAV-ICG-1: TCGGTCCGTCCGTGTGGAGTC (SEQ ID NO 10)
MAV-ICG-22: GTGGCCGGCGTTCATCGAAA (SEQ ID NO 11)
MIN-ICG-1: GCATAGTCCTTAGGGCTGATGCGTT (SEQ ID NO 12)
MIN-ICG-2: GCTGATGCGTTCGTCGAAATGTGTA (SEQ ID NO 13)
MIN-ICG-22: CTGATGCGTTCGTCGAAATGTGT (SEQ ID NO 14)
MIN-ICG-222: TGATGCGTTCGTCGAAATGTGT (SEQ ID NO 15)
MIN-ICG-2222: GGCTGATGCGTTCGTCGAAATGTGTAA (SEQ ID NO 16)
MAL-ICG-1: ACTAGATGAACGCGTAGTCCTTGT (SEQ ID NO 17)
MHEF-ICG-1: TGGACGAAAACCGGGTGCACAA (SEQ ID NO 18)
MAH-ICG-1: GTGTAATTTCTTTTTTAACTCTTGTGTGTAAGTAAGTG (SEQ ID NO 19)
MCO-ICG-11: TGGCCGGCGTGTTCATCGAAA (SEQ ID NO 20)
MTH-ICG-11: GCACTTCAATTGGTGAAGTGCGAGCC (SEQ ID NO 21)
MTH-ICG-2: GCGTGGTCTTCATGGCCGG (SEQ ID NO 22)
MEF-ICG-11: ACGCGTGGTCCTTCGTGG (SEQ ID NO 23)
MSC-ICG-1: TCGGCTCGTTCTGAGTGGTGTC (SEQ ID NO 24)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 77-100 or 108-110, provided said probe hybridizes specifcally to strains from the MAIS complex. The MAIS complex as defined in this invention comprises all strains of M. avium, M. intracellulare and M. scrorulaceum and all strains closely related to the above mentioned species and not clearly belonging to another defined Mycobacterium species. The latter group of strains are defined in this invention as xe2x80x9cMIC strainsxe2x80x9d (M. intracellulare complex).
Preferentially, at least two, three, four, five, six, seven, eight or more of said probes are used simultaneously.
In still another specific embodiment, the invention provides for a method as described above, to detect and identify one or more M. avium and M. paratuberculosis strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MAV-ICG-1: TCGGTCCGTCCGTGTGGAGTC (SEQ ID NO 10)
MAV-ICG-22: GTGGCCGGCGTTCATCGAAA (SEQ ID NO 11)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 77 and 78 provided said probe hybridizes specifically to M. avium or M. paratuberculosis. 
The sequences as represented in SEQ ID NO 77 and 78 are new.
Preferentially, this embodiment uses both probes in combination.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium intracellulare strains and MIC-strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MAI-ICG-1: CAACAGCAAATGATTGCCAGACACAC (SEQ ID NO 6)
MIL-ICG-11: GAGGGGTTCCCGTCTGTAGTG (SEQ ID NO 7)
MIL-ICG-22: TGAGGGGTTCTCGTCTGTAGTG (SEQ ID NO 8)
MAC-ICG-1: CACTCGGTCGATCCGTGTGGA (SEQ ID NO 9)
MIN-ICG-1: GCATAGTCCTTAGGGCTGATGCGTT (SEQ ID NO 12)
MIN-ICG-2: GCTGATGCGTTCGTCGAAATGTGTA (SEQ ID NO 13)
MIN-ICG-22: CTGATGCGTTCGTCGAAATGTGT (SEQ ID NO 14)
MIN-ICG-222: TGATGCGTTCGTCGAAATGTGT (SEQ ID NO 15)
MIN-ICG-2222: GGCTGATGCGTTCGTCGAAATGTGTAA (SEQ ID NO 16)
MAL-ICG-1: ACTAGATGAACGCGTAGTCCTTGT (SEQ ID NO 17)
MAEF-ICG-1: TGGACGAAAACCGGGTGCACAA (SEQ ID NO 18)
MAH-TCG-1: GTGTAATTTCTTTTTTAACTCTTGTGTGTAAGTAAGTG (SEQ ID NO 19)
MCO-ICG-11: TGGCCGGCGTGTTCATCGAAA (SEQ ID NO 20)
MTH-ICG-11: GCACTTCAATTGGTGAAGTGCGAGCC (SEQ ID NO 21)
MTH-ICG-2: GCGTGGTCTTCATGGCCGG (SEQ ID NO 22)
MEF-ICG-11: ACGCGTGGTCCTTCGTGG (SEQ ID NO 23)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 provided said probe hybridizes specifically to M. intracellulare strains and MIC-strains.
The sequences as represented in SEQ ID NO 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 are new.
Preferentially, at least two, three, four, five, six, seven, eight or more of said probes are used simultaneously.
In still another specific embodiment, the invention provides for a method as described above, to detect and identify one or more Mycobacterium intracellulare strains in a sample, wherein step (iii) comprises hybridizing to at least the following probes:
MIN-ICG-1: GCATAGTCCTTAGGGCTGATGCGTT (SEQ ID NO 12)
or to equivalents of said probe,
and/or to any probe derived from SEQ ID NO 89 provided said probe hybridizes specifically to M. intracellulare strains.
In still another specific embodiment, the invention provides for a method as described above, to detect and identify one or more Mycobacterium scrofulaceum strains in a sample, wherein step (iii) comprises hybridizing to the following probe:
MSC-ICG-1: TCGGCTCGTTCTGAGTGGTGTC (SEQ ID NO 24)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 100 provided said probe hybridizes specifically to M. scrofulaceum. 
The sequence as represented in SEQ ID NO 100 is new.
In still another specific embodiment the invention provides for a method as described above to detect and identify one or more Mycobacterium kansasii strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MKA-ICG-1: GATGCGTTTGCTACGGGTAGCGT (SEQ ID NO 25)
MKA-ICG-2: GATGCGTTGCCTACGGGTAGCGT (SEQ ID NO 26)
MKA-ICG-3: ATGCGTTGCCCTACGGGTAGCGT (SEQ ID NO 27)
MKA-ICG-4: CGGGCTCTGTTCGAGAGTTGTC (SEQ ID NO 28)
MKA-ICG-5: CCCTCAGGGATTTTCTGGGTGTTG (SEQ ID NO 182)
MKA-ICG-6: GGACTCGTCCAAGAGTGTTGTCC (SEQ ID NO 183)
MKA-ICG-7: TCGGGCTTGGCCAGAGCTGTT (SEQ ID NO 184)
MKA-ICG-8: GGGTGCGCAACAGCAAGCGA (SEQ ID NO 185)
MKA-ICG-9: GATGCGTTGCCCCTACGGG (SEQ ID NO 186)
MKA-ICG-10: CCCTACGGGTAGCGTGTTCTTTTG (SEQ ID NO 187)
and more preferably to:
MKA-ICG-3: ATGCGTTGCCCTACGGGTAGCGT (SEQ ID NO 27)
MKA-ICG-4: CGGGCTCTGTTCGAGAGTTGTC (SEQ ID NO 28)
MKA-ICG-5: CCCTCAGGGATTTTCTGGGTGTTG (SEQ ID NO 182)
MKA-ICG-6: GGACTCGTCCAAGAGTGTTGTCC (SEQ ID NO 183)
MKA-ICG-7: TCGGGCTTGGCCAGAGCTGTT (SEQ ID NO 184)
MKA-ICG-8: GGGTGCGCAACAGCAAGCGA (SEQ ID NO 185)
MKA-ICG-9: GATGCGTTGCCCCTACGGG (SEQ ID NO 186)
MKA-ICG-10: CCCTACGGGTAGCGTGTTCTTTTG (SEQ ID NO 187)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 101, 167, 168 or 169 provided said probe hybridizes specifically to M. kansasii. 
The sequences as represented in SEQ ID NO 101, 167, 168 and 169 are new.
Preferentially, at least two, three or four of said probes are used simultaneously.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium chelonae strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MCH-ICG-1: GGTGTGGACTTTGACTTCTGAATAG (SEQ ID NO 29)
MCH-ICG-2: CGGCAAAACGTCGGACTGTCA (SEQ ID NO 30)
MCH-ICG-3: GGTGTGGTCCTTGACTATGGATAG (SEQ ID NO 210)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 102, 103 or 174 provided said probe hybridizes specifically to M. chelonae. According to another preferential embodiment, these three probes are used in combination.
The sequences as represented in SEQ ID NO 102, 103 and 174 are new.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium gordonae strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MGO-ICG-1: AACACCCTCGGGTGCTGTCC (SEQ ID NO 31)
MGO-ICG-2: GTATGCGTTGTCGTTCGCGGC (SEQ ID NO 32)
MGO-ICG-5: CGTGAGGGGTCATCGTCTGTAG (SEQ ID NO 33)
and more preferably to:
MGO-ICG-5: CGTGAGGGGTCATCGTCTGTAG (SEQ ID NO 33)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 104, 105 or 106 provided said probe hybridizes specifically to M. gordonae. 
The sequences as represented in SEQ ID NO 104 to 106 are new.
Preferentially, at least two or three of said probes are used simultaneously.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium ulcerans strains or Mycobacterium marinum strains in a sample, wherein step (iii) comprises hybridizing to the following probe:
MUL-MG-1: GGTTTCGGGATGTTGTCCCACC (SEQ ID NO 175)
or to equivalents of said probe,
and/or to any probe derived from SEQ ID NO 157 provided said probe hybridizes specifically to M. ulcerans and M. marinum. 
The sequence as represented in SEQ ID NO 157 is new.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium genavense strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MGV-ICG-1: CGACTGAGGTCGACGTGGTGT (SEQ ID NO 176)
MGV-ICG-2: GGTGTTTGAGCATTGAATAGTGGTTGC (SEQ ID NO 177)
MGV-ICG-3: TCGGGCCGCGTGTTCGTCAAA (SEQ ID NO 211)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 158, 159, 160, 161 or 162 provided said probe hybridizes specifically to M. genavense. 
The sequences as represented in SEQ ID NO 158 to 162 are new.
As described in the examples, M. genavense includes M. genavense strains sensu strictu and a group of closely related snags called M. simiae-like. The former group of strains can be detected specifically with probe MGV-ICG-1 while the latter group hybridizes specifically with probe MGV-ICG-3. Probe MGV-ICG-2 detects both groups.
In still another specific embodiment, the invention provides far a method as described above to detect and identify one or more Mycobacterium xenopi strains in a sample, wherein step (iii) comprises hybridizing to the following probe:
MXE-ICG-1: GTTGGGCAGCAGGCAGTAACC (SEQ ID NO 178)
or to equivalents of said probe,
and/or to any probe derived from SEQ ID NO 163 provided said probe hybridizes specifically to M. xenopi. 
The sequence as represented in SEQ ID NO 163 is new.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterim simiae strains in a sample, wherein step (iii) comprises hybridizing to the following probe:
MSI-ICG-1: CCGGCAACGGTTACGTGTTC (SEQ ID NO 179)
or to equivalents of said probe,
and/or to any probe derived from SEQ ID NO 164 or 165 provided said probe hybridizes specifically to M. simiae. 
The sequence as represented in SEQ ID NO 164 or 165 is new.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium fortuitum strains in a sample. wherein step (iii) comprises hybridizing to at least one of the the following probes:
MFO-ICG-1: TCGTTGGATGGCCTCGCACCT (SEQ ID NO 180)
MPO-ICG-2: ACTTGGCGTGGGATGCGGGAA (SEQ ID NO 181)
or to equivalents of said probes or to any probe derived from SEQ ID NO 166 provided said probe hybridizes specifically to M. fortuitum. 
The sequence as represented in SEQ ID NO 166 is new.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium celatum strains in a sample, wherein step (iii) comprises hybridizing to the following probe:
MCE-ICG-1: TGAGGGAGCCCGTGCCTGTA (SEQ ID NO 190)
or to equivalents of said probe,
and/or to any probe derived from SEQ ID NO 170 provided said probe hybridizes specifically to M. celatum. 
The sequence as represented in SEQ ID NO 170 is new.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium haemophilum strains in a sample, wherein step (iii) comprises hybridizing to the following probe:
MHP-ICG-1: CATGTTGGGCTTGATCGGGTGC (SEQ ID NO 191)
or to equivalents of said probe,
and/or to any probe derived from SEQ ID NO 171, 172 or 173 provided said probe hybridizes specifically to M. haemophilum. 
The sequences as represented in SEQ ID NO 171 to 173 are new.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium malmoense strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MML-ICG-1: CGGATCGATTGAGTGCTTGTCCC (SEQ ID NO 188)
MML-ICG-2: TCTAAATGAACGCACTGCCGATGG (SEQ ID NO 189)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 107 provided said probe hybridizes specifically to M. malmoense. 
The sequence as represented in SEQ ID NO 107 is new.
In still another specific embodiment, the invention provides for a method as described above to detect and identify one or more Mycobacterium strains in a sample, wherein step (iii) comprises hybridizing to at least are of the following probes:
MYC-ICG-1: ACTGGATAGTGGTTGCGAGCATCTA (SEQ ID NO 1)
MYC-ICG-22: CTTCTGAATAGTGGTTGCGAGCATCT (SEQ ID NO 2)
or to equivalents of said probes.
According to a preferred embodiment, both probes are used in combination.
The invention also provides for a method as described above to detect and identify one or more Mycoplasma strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MPN-ICG 1: ATCGGTGGTAAATTAAACCCAAATCCCTGT (SEQ ID NO 49)
MPN-ICG 2: CAGTTCTGAAAGAACATTTCCGCTTCTTTC (SEQ ID NO 50)
MGE-ICG 1: CACCCATTAATTTTTTCGGTGTTAAAACCC (SEQ ID NO 51)
Mycoplasma-ICG: CAAAACTGAAAACGACAATCTTTCTAGTTCC (SEQ ID NO 52)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 124 or 125 provided said probe hybridizes specifically with Mycoplasma species.
Preferentially, at least two, three or four of said probes are used simultaneously.
More particularly, the invention provides for a method as described above to detect and identify one or more Mycoplasma pneumoniae strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
MPN-ICG 1: ATCGGTGGTAAATTAAACCCAAATCCCTGT (SEQ ID NO 49)
MPN-ICG 2: CAGTTCTGAAAGAACATTTCCGCTTCTTTC (SEQ ID NO 50)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 125 provided said probe hybridizes specifically to Mycoplasma pneumoniae. According to a preferred embodiment, both these probes are used in combination.
The sequence as represented in SEQ ID NO 125 is new.
In another particular embodiment, the invention provides for a method as described above to detect and identify one or more Mycoplasma genitalium strains in a sample, wherein step (iii) comprises hybridizing to the following probe:
MGE-ICG 1: CACCCATTAATTTTTTCGGTGTTAAAACCC (SEQ ID NO 51)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 124 provided said probe hybridizes specifically to Mycoplasma genitalium. 
The sequence as represented in SEQ ID NO 124 is new.
The invention also provides for a method as described above to detect and identify one or more Pseudomonas strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
PA-ICG 1: TGGTGTGCTGCGTGATCCGAT (SEQ ID NO 34)
PA-ICG 2: TGAATGTTCGTGGATGAACATTGATT (SEQ ID NO 35)
PA-ICG 3: CACTGGTGATCATTCAAGTCAAG (SEQ ID NO 36)
PA-ICG 4: TGAATGTTCGT(G/A)(G/A)ATGAACATTGATTTCTGGTC (SEQ ID NO 37)
PA-ICG 5: CTCTTTCACTGGTGATCATTCAAGTCAAG (SEQ ID NO 38)
or to equivalent of said probes,
and/or to any probe derived from SEQ ID NO 111, 112, 113, 114 or 115 provided said probe hybridizes specifically to Pseudomonas strains.
The sequences as represented in SEQ ID NO 111 to 115 are new.
Preferentially, at least two, three or four of said probes are used simultaneously.
More particularly, the invention provides for a method as described above to detect and identify one or more Pseudomonas aeruginosa strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
PA-ICG 1: TGGTGTGCTGCGTGATCCGAT (SEQ ID NO 34)
PA-ICG 2: TGAATGTTCGTGGATGAACATTGATT (SEQ ID NO 35)
PA-ICG 3: CACTGGTGATCATTCAAGTCAAG (SEQ ID NO 36)
PA-ICG 4: TGAATGTTCGT(G/A)(G/A)ATGAACATTGATTTCTGGTC (SEQ ID NO 37)
PA-ICG 5: CTCTTFCACTGGTGATCATTCAAGTCAAG (SEQ ID NO 38)
and most preferably to at least one of the following probes:
PA-ICG 1: TGGTGTGCTGCGTGATCCGAT (SEQ ID NO 34)
PA-ICG 4: TGAATGTTCGT(G/A)(G/A)ATGAACATTGATTTCTGGTC (SEQ ID NO 37)
PA-ICG 5: CTCTTTCACTGGTGATCATTCAAGTCAAG (SEQ ID NO 38)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 111 provided said probe hybridizes specifically to Pseudomonas aeruginosa. 
The sequence as represented in SEQ ID NO 111 is new.
Preferentially, at least two, three, four or five of said probes are used simultaneously.
The invention also provides for a method as described above to detect and identify one or more Staphylococcus species in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
STAU-ICG 1: TACCAAGCAAAACCGAGTGAATAAAGAGTT (SEQ ID NO 53)
STAU-ICG 2: CAGAAGATGCGGAATAACGTGAC (SEQ ID NO 54)
STAU-ICG 3: AACGAAGCCGTATGTGAGCATTTGAC (SEQ ID NO 55)
STAU-ICG 4: GAACGTAACTTCATGTTAACGTTTGACTTAT (SEQ ID NO 56)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 139, 140, 141, 142 ,143 or 144 provided said probe hybridizes specifically to Staphylococcus species.
The sequences as represented in SEQ ID NO 139 to 144 are new.
Preferentially, at least two, three or four of said probes are used simultaneously.
More particularly, the invention provides for a method as described above to detect and identify one or more Staphylococcus aureus strains in a sample, wherein step (iii) comprises hybridizing to at least one, and preferably both of the following probes:
STAU-ICG 3: AACGAAGCCGTATGTGAGCATTTGAC (SEQ ID NO 55)
STAU-ICG 4: GAACGTAACTCATGTTAACGTTTGACTTAT (SEQ ID NO 56)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 139, 140, 141, 142 or 143 provided said probe hybridizes specifically to Staphylococcus aureus. According to a preferred embodiment, both these probes are used in combination.
In another specific embodiment the invention provides for a method as described above to detect and identify one or more Staphylococcus epidermidis strains in a sample, wherein step (iii) comprises hybridizing to any probe derived from SEQ ID NO 144 as long as this probe can be caused to hybridize specifically to Staphylococcus epidermidis. 
The invention also provides for a method as described above to detect and identify one or more Acinerobacter strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
ACI-ICG 1: GCTTAAGTGCACAGTGCTCTAAACTGA (SEQ ID NO 57)
ACI-ICG 2: CACGGTAATTAGTGTGATCTGACGAAG (SEQ ID NO 58)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 126, 127, 128, 129 or 130 provided said probe hybridizes specifically to Acinetobacter spp. According to a preferred embodiment, both these probes are used in combination.
The sequences as represented in SEQ ID NO 126 to 130 are new.
More particularly, the invention provides for a method as described above to detect and identify one or more Acinetobacter baumanii strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
ACI-ICG 1: GCTTAAGTGCACAGTGCTCTAAACTGA (SEQ ID NO 57)
ACI-ICG 2: CACGGTAATTAGTGTGATCTGACGAAG (SEQ ID NO 58)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 126 provided said probe hybridizes specifically to Acinetobacter baumanii. According to a preferred embodiment, both these probes are used in combination.
The invention also provides for a method as described above, to detect and identify one or more Listeria strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
LIS-MG 1: CTTTGTAACCGAGAATCATCTGAAAGTGAATC (SEQ ID NO 39)
LMO-ICG 1: AAACAACCTTTACTTCGTAGAAGTAAATTGGTTAAG (SEQ ID NO 40)
LMO-ICG 2: TGAGAGGTTAGTACTTCTCAGTATGTTTGTTC (SEQ ID NO 41)
LMO-ICG 3: AGGCACTATGCTTGAAGCATCGC (SEQ ID NO 42)
LIV-ICG 1: GTTAGCATAAATAGGTAACTATTTATGACACAAGTAAC (SEQ ID NO 43)
LSE-ICG 1: AGTTAGCATAAGTAGTGTAACTATTTATGACACAAG
LISP-ICG 1: CGTTTTCATAAGCGATCGCACGTT (SEQ ID NO 212)
and most preferably to at least one of the following probes:
LIS-ICG 1: CAAGTAACCGAGAATCATCTGAAAGTGAATC (SEQ ID NO 39)
LMO-ICG 3: AGGCACTATGCTTGAAGCATCGC (SEQ ID NO 42)
LISP-ICG 1: CGTTTTCATAAGCGATCGCACGTT (SEQ ID NO 212)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 116, 118, 119, 120, 121, 213, 214 or 215 provided said probe hybridizes specifically to Listeria species.
As described in the examples section, Listeria species encompass Listeria species sensu strictu, and a group of closely related organisms referred to as xe2x80x9cListeria-Like organismsxe2x80x9d. The latter group can be specifically recognized by probe LISP-ICG 1.
The sequences as represented in SEQ ID NO 116, 118 to 121 and 213 to 215 are new.
Preferentially, at least two, three, four, five or six of said probes are used simultaneously.
More particularly, the invention provides for a method as described above, to detect and identity one or more Listeria monocytogenes strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
LMO-ICG 1: AAACAACCTTTACTTCGTAGAAGTAAATTGGTTAAG (SEQ ID NO 40)
LMO-ICG 2: TGAGAGGTTAGTACTCTCAGTATGTTTGTTC (SEQ ID NO 41)
LMO-ICG 3: AGGCACTATGCTTGAAGCATCGC (SEQ ID NO 42)
and most preferably to the following probe:
LMO-ICG 3: AGGCACTATGCTTGAAGCATCGC (SEQ ID NO 42)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 120 provided said probe hybridizes specifically to Listeria monocytogenes. 
Preferentially, at least two, or three of said probes are used simultaneously.
The invention also provides for a method as described above to detect and identify one or more Brucella, strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
BRU-ICG 1: CGTGCCGCCTTCGTTTCTCTTT (SEQ ID NO 59)
BRU-ICG 2: TTCGCTTCGGGGTGGATCTGTG (SEQ ID NO 60)
BRU-ICG 3: GCGTAGTAGCGTTTGCGTCGG (SEQ ID NO 193)
BRU-ICG 4: CGCAAGAAGCTTGCTCAAGCC (SEQ ID NO 194)
and most preferably to at least one of the following probes:
BRU-ICG 2: TTCGCTTCGGGGTGGATCTGTG (SEQ ID NO 60)
BRU-ICG 3: GCGTAGTAGCGTTTGCGTCGG (SEQ ID NO 193)
BRU-ICG 4: CGCAAGAAGCTTGCTCAAGCC (SEQ ID NO 194)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 131, 132 or 154 provided said probe hybridizes specifically to Brucella strains.
The sequences as represented in SEQ ID NO 131, 132 and 154 are stew.
The invention also provides for a method as described above to detect and identity one or more Salmonella strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
SALM-ICG 1: CAAAACTGACTTACGAGTCACGTTTGAG (SEQ ID NO 61)
SALM-ICG 2: GATGTATGCTTCGTTATTCCACGCC (SEQ ID NO 62)
STY-ICG 1: GGTCAAACCTCCAGGGACGCC (SEQ ID NO 63)
SED-ICG 1: GCGGTAATGTGTGAAAGCGTTGCC (SEQ ID NO 64)
and most preferably to the following probe:
SALM-ICG 1: CAAAACTGACTTACGAGTCACGTTTGAG (SEQ ID NO 61)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 133, 134, 135, 136, 137 or 138 provided said probe hybridizes specifically to Salmonella strains.
The sequences as represented in SEQ ID NO 133 to 138 are new.
Preferentially, at least two, three, or four of said probes are used simultaneously.
The invention also relates to a method as described above to detect and identify one or more Chlamydia strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
CHTR-ICG 1: GGAAGAAGCCTGAGAAGGTTTCTGAC (SEQ ID NO 45)
CHTR-ICG 2: GCATTTATATGTAAGAGCAAGCATTCTATTTCA (SEQ ID NO 46)
CHTR-ICG 3: GAGTAGCGTGGTGAGGACGAGA (SEQ ID NO 47)
CHTR-ICG 4: GAGTAGCGCGGTGAGGACGAGA (SEQ ID NO 201)
CHPS-ICG 1: GGATAACTGTCTTAGGACGGTTTGAC (SEQ ID NO 48)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 122, 123 or 197 provided that said probe hybridizes specifically to Chlamydia strains.
Preferentially, at least two, three, four or five of said probes are used simultaneously.
More particularly, the invention relates to a method as described above to detect and identify one or more Chlamydia trachomatis strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
CHTR-ICG 1: GGAAGAAGCCTGAGAAGGTTTCTGAC (SEQ ID NO 45)
CHTR-ICG 2: GCATTTATATGTAAGAGCAAGCATTCTATTTCA (SEQ ID NO 46)
CHTR-ICG 3: GAGTAGCGTGGTGAGGACGAGA (SEQ ID NO 47)
CHTR-ICG 4: GAGTAGCGCGGTGAGCACGAGA (SEQ ID NO 201)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 123 or 197 provided said probe hybridizes specifically to Chlamydia trachomatis. 
The sequences as represented in SEQ ID NO 123 and 197 are new,
Preferentially, at least two, three or four of said probes are used simultaneously.
In another particular embodiment, the invention provides for a method as described above to detect and identify one or more Chlamydia psittaci strains in a sample, wherein step (iii) comprises hybridizing to at least the following probe:
CHPS-ICG 1: GGATAACTGTCTTAGGACGGTTTGAC (SEQ ID NO 48)
or to equivalents of said probe,
and/or to any probe derived from SEQ ID NO 122 provided said probe hybridizes specifically to Chlamydia psittaci. 
The sequence of SEQ ID NO 122 is new,
The invention also provides for a method as described above, to detect one or mare Streptococcus strains in a sample, wherein step (iii) comprises hybridizing to any probe derived from SEQ ID NO 145, 146, 147, 148, 149, 150, 151, 152 or 153 provided said probe hybridizes specifically to Streptococcus strains, or equivalents of these probes.
The sequences as represented in SEQ ID NO 145, 146, 147, 148, 149, 150, 151, 152 or 153 are new.
The invention also provides for a method as described above, to detect one or more Yersinia enterocolitica strains in a sample, wherein step (iii) comprises hybridizing to at least one of the following probes:
YEC-ICG 1: GGAAAAGGTACTGCACGTGACTG (SEQ ID NO 198)
YEC-ICG 2: GACAGCTGAAACTTATCCCTCCG (SEQ ID NO 199)
YEC-ICG 3: GCTACCTGTTGATGTAATGAGTCAC (SEQ ID NO 200)
or to equivalents of said probes,
and/or to any probe derived from SEQ ID NO 195 or 196, provided said probe hybridize specifically to Yersinia enterocolitica. 
The sequences as represented in SEQ ID NO 195 and 196 are new.
In some cases it may be advantageous to amplify not all organisms present in a sample, but only more specific taxa, which are considered to be relevant. In these cases the invention provides for primers allowing the specific amplification of the spacer region for only those beforehand defined taxa.
The invention thus provides for a method as described above to detect and identify specifically Chlamydia trachomatis is a sample, wherein step (ii) comprises amplification of the 16S-23S rRNA spacer region or a part of it, using at least one of the following primers:
CHTR-P1: AAGGTTTCTGACTAGGTTGGGC (SEQ ID NO 69)
CHTR-P2: GGTGAAGTGCTTGCATGGATCT (SEQ ID NO 70)
or equivalents of these primers, said equivalents differing in sequences from the above mentioned primers by changing one or more nucleotides, provided that said equivalents still amplify specifically the spacer region or part of it from Chlamydia trachomatis. 
Preferably both primers are used.
The invention also provides for a method as described above to detect and identify specifically Listeria species in a sample, wherein step (ii) comprises amplification of the 16S-23S rRNA spacer region or a part of it, using at least one of the following primers:
LIS-P1: ACCTGTGAGTTTTCGTTCTTCTC (SEQ ID NO 71)
LIS-P2: CTATTTGTTCAGTTTTGAGAGGTT (SEQ ID NO 72)
LIS-P3: ATTTTCCGTATCAGCGATGATAC (SEQ ID NO 73)
LIS-P4: ACGAAGTAAAGGTTGTTTTPCT (SEQ ID NO 74)
LIS-P5: GAGAGGTTACTCTCTTTTATGTCAG (SEQ ID NO 75)
LIS-P6: CTTTTATGTCAGATAAAGTATGCAA (SEQ ID NO 202)
LIS-P7: CGTAAAAGGGTATGATTATTTG (SEQ ID NO 203)
or equivalents of these primers, said equivalents differing in sequence from the above mentioned primers by changing one or more nucleotides, provided that said equivalents still amplify specifically the spacer region or part of it from Listeria species.
The invention also relates to a method as described above to detect and identify specifically Mycobacterium species in a sample, wherein step (ii) comprises amplification of the 16S-23S rRNA spacer region or a part of it, using at least one of the following primers:
MYC-P1: TCCCTTGTGGCCTGTGTG (SEQ ID NO 65)
MYC-P2: TCCTTCATCGGCTCTCGA (SEQ ID NO 66)
MYC-P3: GATGCCAAGGCATCCACC (SEQ ID NO 67)
MYC-P4: CCTCCCACGTCCTTCATCG (SEQ ID NO 68)
MYC-P5: CCTGGGTTTGACATGCACAG (SEQ ID NO 192)
or equivalents of these primers, said equivalents differing in sequence from the above mentioned primers by changing one or more nucleotides, provided that said equivalents still amplify specifically the spar region or part of it from Mycobacterium species.
The invention also provides for a method as described above to detect and identify specifically Brucella species in a sample, wherein step (ii) comprises amplification of the 16S-23S rRNA spacer region or part of it, using at least one of the following primers:
BRU-P1: TCGAGAATTGGAAAGACGTC (SEQ ID NO 204)
BRU-P2: AAGAGGTCGGATTTATCCG (SEQ ID NO 205)
BRU-P3: TTCGACTCCAAATGCTCG (SEQ ID NO 206)
BRU-P4: TCTTAAAGCCGCATTATGC (SEQ ID NO 207)
or equivalents of these primers, said equivalents differing in sequence from the above-mentioned primers by changing one or more nucleotides, provided that said equivalents still amplify specifically the spacer region of part of it from Brucella species.
The invention also provides for a method as described above to detect and identify specifically Yersinia enterocolitica species in a sample, wherein step (ii) comprises amplification of the 16S-23S rRNA spacer region or part of it, using at least one of the following primers:
YEC-P1: CCTAATGATATTGATTCGCG (SEQ ID NO 208)
YEC-P2: ATGACAGGTTAATCCTTACCCC (SEQ ID NO 209)
or equivalents of these primers, said equivalents differing in sequence from the above-mentioned primers by changing one or more nucleotides, provided that said equivalents still amplify specifically the spacer region of part of it from Yersinia enterocolitica species.
The invention also provides for a composition comprising at least one of the probes and/or primers as defined above.
Said composition may comprise any carrier, support, label or diluent known in the art for probes or primers, more particularly any of the labels or supports detailed in the definitions section.
The invention relates more particularly to isolated probes and primers as defined above, more particularly any of the probes as specified in Table 1a or any of the primers as specified in Table 1b.
According to another embodiment, the present invention relates also to new spacer region sequences as defined above and as set out in FIGS. 1-103 (SEQ ID NO 76 to 154, SEQ ID NO 157 to 174, SEQ ID NO 195 to 197 and SEQ ID NO 213 to 215).
In another embodiment the invention provides for a reverse hybridization method comprising any of the probes as defined above, wherein said probes are immobilized on a known location on a solid support, more preferably on a membrane strip.
In yet another embodiment the invention provides for a kit for the detection and identification of at least one micro-organism, or the simultaneous detection and identification of several micro-organisms in a sample, comprising the following components:
(i) when appropriate, at least one suitable primer pair to allow amplification of the intercistronic 16S-23S rRNA spacer region, or a part of it;
(ii) at least one of the probes as defined above;
(iii) a buffer, or components necessary to produce the buffer, enabling a hybridization reaction between said probes and the polynucleic acids present in the sample, or the amplified products thereof;
(iv) a solution, or components necessary to produce the solution, enabling washing of the hybrids formed under the appropriate wash conditions;
(v) when appropriate, a means for detecting the hybrids resulting from the preceding hybridization.